Mcw: Molecules To Cells, Block 1

Front 1

In the "native structure" of a protein, where are the hydrophobic residues found? Hydrophilic residues?

Back 1

- Hydrophobic residues are mostly buried in the interior
- Hydrophilic residues are mostly on the surface

Front 2

Which of the amino acid residues is NOT achiral / optically inactive? Why?

Back 2

- Glycine
- It's α-Carbon is attached to 2 H atoms

Front 3

What are the two mirror image forms of a chiral amino acid called?

Back 3

Enantiomers

Front 4

Which configuration are all amino acids (except glycine) found in mammalian proteins?

Back 4

L-configuration

Front 5

How do you draw an amino acid in the "L-configuration"?

Back 5

- α-C in center
- NH3+ wedged to left
- COO- dashed to top
- H wedged to right
- R group dashed down

Front 6

What are the two categories of amino acids and the two sub-categories for each?

Back 6

Hydrophobic / Apolar:
- Aliphatic
- Aromatic
Hydrophilic / Polar:
- Uncharged
- Charged

Front 7

Which amino acids are categorized as hydrophobic/apolar and aliphatic?

Back 7

- Glycine (Gly, G)
- Alanine (Ala, A)
- Valine (Val, V)
- Leucine (Leu, L)
- Isoleucine (Ile, I)
- Methionine (Met, M)
- Proline (Pro, P)

Front 8

What is unique about the amino acid Proline (Pro, P)?

Back 8

- Proline has an imino group (2 N-C bonds)
- All other amino acids have an amino group (1 N-C bond)

Front 9

Which amino acids are categorized as hydrophobic/apolar and aromatic?

Back 9

- Phenylalanine (Phe, F)
- Tyrosine (Tyr, Y)
- Tryptophan (Trp, W)

Front 10

How can protein concentration be estimated?

Back 10

Measuring the absorbance at 280 nm; aromatic side chains (Trp, Tyr, Phe) have characteristic UV absorbance at 280nm

Front 11

Which amino acids are categorized as hydrophilic/polar and uncharged?

Back 11

- Serine (Ser, S)
- Threonine (Thr, T)
- Asparagine (Asn, N)
- Glutamine (Gln, Q)
- Cysteine (Cys, C)

Front 12

Which amino acids are categorized as hydrophilic/polar and charged? What charges do these residues have?

Back 12

- Lysine (Lys, K) +
- Arginine (Arg, R) +
- Histidine (His, H) +
- Aspartate (Asp, D) -
- Glutamate (Glu, E) -

Front 13

Positively charged residues are acidic or basic? Which residues?

Back 13

Basic: Lys, Arg, His

Front 14

Negatively charged residues are acidic or basic? Which residues?

Back 14

Acidic: Asp, Glu

Front 15

What does the pKa of a group tell you?

Back 15

The pKa is the pH at which it is 50% protonated and 50% unprotonated

Front 16

When the pH is LOWER than the pKa, what is the status of that group?

Back 16

- Higher concentration of H+ in solution
- >50% of group is protonated

Front 17

When the pH is HIGHER than the pKa, what is the status of that group?

Back 17

- Lower concentration of H+ in solution
- <50% of group is protonated

Front 18

What equation can be used to determine the ratio of protonated (HA) to unprotonated (A-) forms?

Back 18

Henderson-Hasselbalch Equation:
pH = pKa + log[A-]/[HA]

Front 19

What does the isoelectric point, pI represent?

Back 19

An intermediate pH characteristic of the amino acid, peptide or protein in which the net charge is zero

Front 20

What does it mean for the pH to be "buffered"?

Back 20

When the pH is near the pKa of the residue/protein it takes significantly more acid or base to change the pH than when further from the pKa; therefore the solution is STABILIZED near the pKa values of the ionizable groups

Front 21

Which important molecules in the body help "buffer" the intracellular and extracellular fluids to keep cells, tissues, and organisms surviving?

Back 21

- Amino acids, peptides, proteins
- Phosphate
- Bicarbonate

Front 22

In what type of proteins are disulfide linkages common? Where are they uncommon?

Back 22

- Common: extracellular proteins
- Uncommon: intracellular proteins

Front 23

Which hormone is highly dependent upon the increased conformational stability due to disulfide bonds?

Back 23

Insulin (contains 3 disulfide bonds)

Front 24

What features of the peptide bond limit the number of conformational states possible for a polypeptide?

Back 24

1. Strong electronegativity of C=O relative to amide N leads to resonance structures; prevents bond rotation around the amid bond
2. Trans conformation of R groups is preferred due to lower energy / more stable / less steric hindrance

Front 25

Simply, what determines the protein's folding and 3D structure?

Back 25

The sequence of amino acids in a protein (the primary structure)

Front 26

What are the two types of secondary structures of proteins?

Back 26

- Right-handed α-helix
- β-pleated sheet (parallel or antiparallel or mixed)

Front 27

What is it called when secondary structures make stable associations with one another?

Back 27

Super-secondary structures

Front 28

What is a domain?

Back 28

A structure above super-secondary structures that is the smallest thermodynamically stable unit of protein structure; often accounts for part of an enzyme's active site or may catalyze one part of a complex reaction sequence

Front 29

What does the tertiary structure represent? How many domains are in a tertiary structure?

Back 29

- Final thermodynamically stable structure of a single polypeptide chain
- One or more domains (often 2 or 3)

Front 30

When multiple polypeptide chains come together to form a globular protein, this level of structure is called what?

Back 30

Quaternary structure

Front 31

What are three examples of fibrous proteins we need to know about?

Back 31

- α-Keratin
- Collagen
- Elastin

Front 32

What is α-Keratin a primary component of?

Back 32

Hair and nails

Front 33

What is the structure of α-Keratin?

Back 33

- Two right handed α-helices
- α-helices are intertwined to form a left-handed supercoil called an α-coiled coil
- These can bond non-covalently (H-bonds, hydrophobic intxns, and ionic bonds) and covalently (disulfide bonds)

Front 34

What distinguishes the α-Keratin found in hair with that which is found in nails?

Back 34

- α-Keratin in hair has a moderate number of disulfide bonds
- α-Keratin in nails has a high number of disulfide linkages to make it rigid

Front 35

What is the most abundant protein in the body?

Back 35

Collagen (20-25% of total protein)

Front 36

What is the structure of Collagen?

Back 36

- Three collagen chains -Gly-X-Y-(X often proline, Y often hydroxyproline or hydroxylysine), called α-chains, are left-handed helices
- These three are wrapped around each other in a right-handed triple helix
- Multiple triple helices interdigitate to form collagen microfibrils

Front 37

What amino acids are important to the structure of Collagen? Why?

Back 37

- Proline - stack long outside of helix providing rigidity
- Glycine - found internally in the helix due to small R group (every third residue)

Front 38

Which amino acids are often found on the outside of the triple helix of Collagen? Why is this significant?

Back 38

- Proline, hydroxyproline and hydroxylysine
- These form bonds between neighboring chains resulting in aggregation into long fibers

Front 39

Why is Vitamin C important for Collagen formation?

Back 39

- Vitamin C / Ascorbate is required to catalyze the hydroxylation reactions to form hydroxyproline and hydroxylysine
- These residues are important for stabilizing the triple-helical structure

Front 40

If there is a vitamin C / ascorbate deficiency, what can happen?

Back 40

Scurvy (symptoms include bleeding gums) due to decreased tensile strength of collagen

Front 41

Where is the protein elastin commonly found?

Back 41

Connective tissue: lungs, large arterial walls, elastic ligaments

Front 42

What amino acids predominantly make up elastin?

Back 42

- Small, non-polar amino acids (e.g., glycine, alanine, and valine)
- Proline and lysine

Front 43

Which connective tissue protein has rubber-like properties?

Back 43

Elastin

Front 44

What does the enzyme elastase do? What can inhibit elastase?

Back 44

- Elastase is an enzyme inhibitor of elastin (which can destroy the alveolar epithelium)
- Elastase is inhibited by α1-antitrypsin

Front 45

What clinical problem can occur if there is not enough of α1-antitrypsin (elastase inhibitor)?

Back 45

Emphysema (due to the destruction of the alveolar epithelium)

Front 46

What is a gene family?

Back 46

- Similar genes formed by duplication of an original gene; the genes have similar biochemical functions
- Hemoglobin has two gene families, α-globin and β-globin

Front 47

What happens within a gene family during development? Specifically with globins?

Back 47

- The expression of members of a gene family change during development
- Composition of the Hb tetramer changes with gestational and post-partum age
- I.e., ζ (zeta) α-globin is expressed briefly embryonically and then only α is expressed throughout fetal growth and adulthood
- I.e., ε (epsilon) β-globin is expressed embryonically, followed by γ (gamma) during fetal growth and later by β

Front 48

How does the structure of myoglobin compare to hemoglobin?

Back 48

- Myoglobin is a monomeric protein
- Hemoglobin is a heterotetrameric protein (αβ)2
- The α and β subunits are similar sequentially and structurally and evolutionarily related to myoglobin
- Both contain a Fe2+-protoporphyrin IX prosthetic group that binds O2

Front 49

How does the function of myoglobin compare to hemoglobin?

Back 49

- Hemoglobin transports O2 from the lungs to peripheral tissues
- Myoglobin is found in muscle and is designed to store O2

Front 50

Why is it necessary for hemoglobin to demonstrate cooperativity of oxygen binding?

Back 50

- When Hb is in the muscles (resting or working) the pO2 is much lower (due to the consumption of O2)...this leads to Hb releasing its O2
- In the lungs, the pO2 is higher, which leads to increased binding of O2 to Hb

Front 51

How does hemoglobin demonstrate cooperativity of oxygen binding?

Back 51

- Binding of a single oxygen to one Hb subunit induces a conformation change that is partially transmitted to adjacent subunits
- This induces an increased affinity for oxygen
- As more oxygens bind to subunits the affinity is increased and the molecule transitions from the T (tense) state to the R (relaxed) state

Front 52

In general, how do H+, CO2, and 2,3-DPG regulate O2 binding to hemoglobin?

Back 52

All act as allosteric regulators/effectors, which means they bind at a site other than the active site to induce a conformational change that reduces the Hb's affinity for O2

Front 53

Specifically, how does [H+] regulate O2 binding to hemoglobin?

Back 53

- As pH lowers (or [H+] increases), the affinity of Hb for O2 decreases
- This leads to an increase in release of O2 in the tissues that need it
- Catabolism can continue
- H+ binding results from a shift in pKa of specific residues

Front 54

Specifically, how does [CO2] regulate O2 binding to hemoglobin?

Back 54

- As [CO2] increases, there is a lower affinity for O2
- CO2 builds up in tissues that are actively catabolizing fuel molecules, therefore more O2 is required to continue working

Front 55

What kind of effectors / regulators are H+ and CO2?

Back 55

Heterotropic Negative Allosteric Effectors
- Heterotropic because they are not O2
- Negative because they decrease affinity for O2
- Allosteric because they bind to a site other than that which O2 binds to

Front 56

Specifically, how does 2,3-DPG regulate O2 binding to hemoglobin?

Back 56

- 2,3-DPG is a negative allosteric effector
- It binds to a cleft between the β subunits
- Binding is stabilized by ionic intxns
- RBCs have regulatory mechanisms to control the conc. of 2,3-DPG so as to fine tune the affinity of Hb for O2

Front 57

How does O2 affect O2 binding to hemoglobin?

Back 57

- O2 is a positive homotropic allosteric effector of O2 binding
- It is a negative allosteric effector of H+ and CO2 binding
- This reciprocal relationship between O2 and H+ is termed the Bohr effect or isohydric shift

Front 58

What is the Bohr effect (aka isohydric shift)?

Back 58

- O2 is a negative allosteric effector of H+
- H+ is a negative allosteric effector of O2
- This reciprocal relationship between O2 and H+ is called the Bohr Effect

Front 59

What is the molecular basis of sickle cell anemia?

Back 59

- Homozygous recessive disease
- Caused by a point mutation in adult β-globulin gene, leads to substitution of Valine (Val) from Glutamic Acid (Glu)
- Val is hydrophobic (Glu is hydrophilic) and creates a sticky patch on deoxyHb that leads to polymerization of Hb tetramers
- This leads to the sickle cell shape which often blocks microcirculation as well as a shorter half-life

Front 60

What are the four common forms of Sickle Cell Disease (SCD)?

Back 60

- Homozygous HbS disease (HbSS)
- HbS/βo thalassemia
- Hbs/β+ thalassemia
- HbSC

Front 61

The "S" mutation of Hb causes what?

Back 61

- Glu6 --> Val
- Glu is hydrophilic, Val is hydrophobic
- Val creates sticky patch on deoxyHb; leads to polymerization of Hb tetramers
- Leads to sickled shape and reduced deformability of RBCs

Front 62

The "C" mutation of Hb causes what?

Back 62

- Glu --> Lys
- Negative to positive charge
- Interacts with membrane causing formation / sickling problems

Front 63

The βo thalassemia mutation leads to what in Hb?

Back 63

- Lack of β chain
- Still have two α chains

Front 64

The β+ thalassemia mutation leads to what in Hb?

Back 64

- Reduced β chain
- RBC are smaller due to less Hb

Front 65

What are common complications of SCD?

Back 65

- Vaso-occlusive crises
- Hemolysis and anemia
- Ischemia
- Chronic pain
- Dactylitis - painful/swelling of hands and feet (infants)
- Frequent infections (spleen is busy metabolizing RBC and is less capable of fighting infections)
- Delayed growth
- Vision problems
- Chest pain
- Renal problems
- Pulmonary hypertension
- Necrosis of bones

Front 66

What components make up a nucleic acid?

Back 66

- Nitrogenous base (purines and pyrimidines)
- Ribose or deoxyribose
- Phosphate group

Front 67

What are the different nitrogenous bases and how are they categorized? How many rings does each kind have?

Back 67

- Purine: Adenine, Guanine (2 rings)
- Pyrimidine: Cytosine, Uracil, Thymine (1 ring)

Front 68

How can you distinguish between the purines, adenine and guanine?

Back 68

- Both have two rings
- Guanine has a carbonyl carbon on the 6 membered ring and an amino (NH2) group
- Adenine has only an NH2 group on the 6 membered ring (no carbonyl)

Front 69

How can you distinguish between the pyrimidines, cytosine, uracil and thymine?

Back 69

- All have one ring
- Cytosine has a carbonyl and an amino (NH2) group
- Uracil has two carbonyl groups
- Thymine has two carbonyl groups and a methyl group

Front 70

How do you distinguish between the two nucleic acid sugars, deoxyribose and ribose?

Back 70

- Both have OH groups at 1', 3' and 5' positions
- Deoxyribose has an H at the 2' position (total of 3 OH groups)
- Ribose has an OH group at the 2' position (total of 4 OH groups)

Front 71

What's the difference between a nucleoTide and a nucleoSide?

Back 71

- NucleoTides have a base, sugar AND a phosphate
- NucleoSides have ONLY a base and a sugar

Front 72

How are the components of a nucleotide connected?

Back 72

- Sugar attaches to phosphates at 5' OH and 3' OH via phosphodiester bridge (backbone)
- Sugar attaches to the base at the 1' position

Front 73

How do DNA and RNA differ?

Back 73

- DNA uses the sugar deoxyribose
- RNA uses the sugar ribose
- DNA uses the base thymine
- RNA uses the base uracil

Front 74

Describe the structure of double-stranded DNA.

Back 74

- Right-handed, B-form helix is most common
-Major groove (12A) and minor groove (6A)
- Complementary strands run antiparallel to each other
- Bases are on inside of helix with bases stacked perpendicular to helix axis (promotes stability)
- Repeats after 10.5 residues / 36A
- Held together by hydrogen base pairing (AT and CG)

Front 75

How many hydrogen base pairs keep adenine and thymine/uracil together?

Back 75

Two hydrogen bonds

Front 76

How many hydrogen base pairs keep guanine and cytosine together?

Back 76

Three Hydrogen bonds

Front 77

What are the three forms of DNA helices?

Back 77

- A form = more symmetrical right handed helix
- B form = major and minor grooves; right handed; most common
- Z form = left handed helix

Front 78

What are two ways to separate a double helix by disrupting hydrogen bonds?

Back 78

Changes in pH and temperature

Front 79

How does ionic composition affect denaturation of DNA?

Back 79

- Ionic composition (salt concentration) effects denaturing and annealing RATES

Front 80

How can the denaturation and renaturation of DNA be measured?

Back 80

- Spectrophotometrically at 260 nm
- Single strands absorb better (free to absorb light)
- Double stranded DNA doesn't absorb as well because the stacking of bases blocks the UV light from absorption

Front 81

If a DNA helix is being denatured, how will the absorbance at 260 nm be affected?

Back 81

There will be an increase in absorbance due to less base stacking

Front 82

If a DNA helix is being renatured, how will the absorbance at 260 nm be affected?

Back 82

There will be a decrease in absorbance due to increased base stacking

Front 83

What is the melting temperature (Tm) of DNA? What does it depend upon?

Back 83

- Defined as the temperature at which half (50%) of the helical structure is lost
- Depends on % GC content
- Also affected by ionic strength

Front 84

How is the Tm affected by a higher GC content?

Back 84

The Tm will be reached at a higher temperature because GC base pairs have 3 H bonds vs 2 H bonds for AT pairs

Front 85

How is the Tm affected by ionic strength? Explain.

Back 85

- High salt favors duplex
- Low salt favors denatured
- Electrostatic repulsion caused by charged phosphate backbone; if there is more salt to neutralize those charges then the DNA strands will be less repelled by each other

Front 86

How do linear and circular DNA differ?

Back 86

- Circular can be relaxed or supercoiled due to the nature of twists adding tension
- Linear can feel some tension due to twisting if ends are anchored by proteins

Front 87

Most DNA is negatively or positively supercoiled?

Back 87

Negatively supercoiled; may aid in transcription and replication

Front 88

What is the function of topoisomerases?

Back 88

- Enzymes that change the topological state of circular DNA but not its covalent state
- Two types

Front 89

Compare and contrast the differences between Type 1 and Type 2 Topoisomerases.

Back 89

- Time 1: create transient single-stranded breaks in DNA; nicking-closing enzymes; can relax both negatively and positively supercoiled DNA; removes 1 twist at a time
- Type 2: create transient double-stranded breaks; requires ATP to complete cycle; removes 2 twists at a time

Front 90

What are the steps by which Type 1 Topoisomerases function?

Back 90

- Cleave on strand of DNA
- Pass a segment of DNA through the break
- Reseal the break
- (NO energy required)

Front 91

What are the steps by which Type 2 Topoisomerases function?

Back 91

- Two strands of DNA are cleaved (ATP required)
- DNA is passed through break
- Break is resealed

Front 92

When is the function of topoisomerases necessary?

Back 92

- When unzipping DNA twists are added to remaining helix
- Topoisomerases are important to reduce the tension

Front 93

What drugs inhibit E.coli DNA gyrase (type 2 topoisomerase)? What are these drugs used to target?

Back 93

- Nalidixic acid and Ciprofloxacin (quinolones)
- By only targeting bacteria they make for effective antibiotics
- Ciprofloxacin is used for anthrax

Front 94

What drugs inhibit eukaryotic Type II topoisomerases? What are these drugs used to target?

Back 94

- Etoposide and doxorubicin
- Used in cancer chemotherapy
- Specifically inhibit the Topo ATPase activity (increases rate of cleaving but reduces rate of resealing); this results in the arrest of DNA replication and RNA transcription
- Cancer cells are rapidly replicating and have higher levels of Type II topoisomerases making these drugs lethal to these cells and less damaging to slow-growing cells

Front 95

What is the general hierarchy of chromatin packing?

Back 95

- 1st order: nucleosome
- 2nd order: 30-nanometer fiber
- 3rd order: chromatin loop
- 4th order: miniband
- 5th order: chromosome

Front 96

Describe the first order of chromatin structure.

Back 96

- Nucleosome consists of ~200bp of DNA wrapped around octamer of core histone proteins
- Covalent modifications of core histones determine transcriptional activity of packaged genes

Front 97

Describe the second order of chromatin structure.

Back 97

- 30-nanometer fiber
- Six 200bp nucleosomes packaged in a solenoid arrangement into one 1200bp 30nm fiber
- Binding of linker histone is required to stabilize 30nm fiber

Front 98

Describe the third order of chromatin structure.

Back 98

- Chromatin loop
- Fifty 1200bp 30nm fibers form loops of chromatin containing ~60,000bp DNA
- Each loop is attached to the nuclear matrix at the base
- Contains one to several genes

Front 99

Describe the fourth order of chromatin structure.

Back 99

- Miniband
- Consists of a tandem of 18 chromatin loops = ~1 million bp DNA
- Encircles axis of mitotic chromosome

Front 100

Describe the fifth order of chromatin structure.

Back 100

- Chromosome
- Contains a single linear DNA molecule, packaging an average of 75 million bp of DNA (75 x minibands stacked on top of one another)
- Includes a centromere and two telomeres, loci, or repetitive DNA located in the middle and at ends of chromosome, respectively

Front 101

What is "Chromatin"?

Back 101

- Genomic DNA
- Nuclear proteins

Front 102

What is the difference between "euchromatin" and "heterochromatin"?

Back 102

- Heterochromatin - 10% of chromatin in typical cell; highly condensed and usually transcriptionally inactive
- Euchromatin - 90% of chromatin in typical cell; less condensed and usually transcriptionally active

Front 103

Which level of the hierarchy of chromatin structure is related to looking like "beads on a string"? What makes up the "beads" and the "string"?

Back 103

- 1st order: nucleosome
- Beads = nucleosome (half protein - octamer of histone and a few non-histones/half DNA)
- String = linkers (H1 histones)

Front 104

What histones are found in a nucleosome?

Back 104

- Two of each core histone:
- H2A
- H2B
- H3
- H4
- Form an octamer

Front 105

Are histones acidic or basic? Which residues make them this way?

Back 105

Basic; lots of lysine+ and arginine+

Front 106

How can we know that histones are vital cellularly?

Back 106

Highly conserved sequences

Front 107

What is the function of histones?

Back 107

- Structural role (wrapping DNA for packing)
- Inhibition of transcription

Front 108

What is necessary for transcription of a gene to occur (re: histones)?

Back 108

- Histones must be modified via acetylation of the Lysine+ and Arginine+ residues
- This neutralizes the positive charges
- Results in repulsion of histones from DNA gene sites causing chromatin "opening" allowing in transcriptional machinery

Front 109

How can histones be modified to inhibit transcription?

Back 109

- Methylation of Histone H3 causes formation of the non-transcribable "hetero"-chromatin
- OR Phosphorylation of histones usually repressed transcription by preventing the condensation chromatin into recognizable chromosomes as cell enters M-phase (mitosis)

Front 110

What is meant by the term "non-histone"?

Back 110

- Applies to any chromatin protein that is not a histone
- Includes DNA repair enzymes, DNA and RNA polymerases, and transcription factors

Front 111

What are all of the ways in which histones can be modified? Which residues are impacted directly by these modifications?

Back 111

- Acetylation - lysine
- Methylation - lysine and arginine
- Phosphorylation - serine and threonine
- Ubiquitination - lysine
- Sumoylation - lysine
- Usually affects residues on amino-terminal that protrude from nucleosome

Front 112

By which distinct mechanisms do histone modifications affect chromosome function?

Back 112

- Effects on electrostatic charge of histone and resulting changes in conformation (acetylation/deacetylation)
- Modifications which provide binding sites for regulatory proteins containing protein domains that recognize these modifications

Front 113

What are epigenetic modifications?

Back 113

Heritable changes in gene function that occur without a change in the sequence of the DNA

Front 114

Functionally how does DNA methylation by DNA methyltransferases (DNMTs) affect gene expression?

Back 114

DNA methylation represses gene expression by blocking binding of transcription factors that normally activate gene expression

Front 115

Structurally how does DNA methylation by DNA methyltransferases (DNMTs) affect gene expression?

Back 115

- Methylated CpG in DNA is recognized and bound by methyl CpG binding proteins (MBPs)
- MBPs recruit histone deacetylases (HDACs)
- HDACs carry out histone deacetylation
- Histones in de-acetylated state bind DNA more tightly which inhibits gene transcription by blocking binding of transcription activator proteins
- Additionally MBPs can recruit histone methyltransferases (HMTs)
- Methylated histones are recognized and bound by HP1 protein, completely silencing gene expression

Front 116

How can epigenetic modifications directly affect cancer?

Back 116

- These epigenetic modifications (methylation or deacetylation) may lead to the suppression of expression of tumor suppressor genes
- Leads to high rate of uncontrolled cell growth

Front 117

What is the function of tumor suppressor genes?

Back 117

- Help control cell growth by regulating the processes of mitosis and cell division

Front 118

What is the role of Histone H1?

Back 118

- Acts as the linker protein for the first level of chromatin packing (connects nucleosomes)
- In second order packing, it participates in packaging the 6-200bp nucleosomes into one-1200bp 30nm fiber

Front 119

Which order of chromatin packing looks like a solenoid?

Back 119

Second order: 30nm fiber

Front 120

What is the nuclear matrix? Which order of chromatin hierarchy is this an important aspect of?

Back 120

- Nuclear matrix is a proteinaceous structure that contains structural and gene regulatory machinery
- Important for the 3rd order: loops which attach to it

Front 121

At what order of chromatin hierarchy does "supercoiling" become possible?

Back 121

- 3rd order: loops
- By being bound to the nuclear matrix, the DNA can become supercoiled; effectively circularizes the DNA

Front 122

Besides conferring supercoiling abilities at the 3rd order of chromatin hierarchy (loops), what other advantages does this level confer?

Back 122

Genes located in one loop can interact with up- and downstream regulatory sequences (e.g., enhancers) in an organized way

Front 123

How do "loops" (3rd order) get organized into "minibands" (4th order)?

Back 123

- Nuclear matrix is in the center with 18 loops spanning out around the chromosome axis
- Looks like flower petals from above
- Proximity of loops on adjacent minibands would permit regulatory sequences in distal loops to interact

Front 124

Hyper-phosphorylation of chromatin proteins (especially histone H1) permits what?

Back 124

Hyper-condensation of chromatin into mitotic chromosomes (5th order)

Front 125

What are the features of the mitotic chromosome?

Back 125

- Centromere at center
- Two telomeres at either end (repetitive DNA caps; GGGTTA for up to ~15,000 bp)

Front 126

As telomeres get shorter in somatic cells with each cell division, what term does this lead to?

Back 126

Cellular Senescence (deterioration with age)

Front 127

How are telomeres maintained in germ-line cells?

Back 127

Telomerase maintains telomere length after cell division

Front 128

Why is it important for telomerase to be inactive in somatic cells?

Back 128

- If mutated and telomerase is activated it can lead to abnormal maintenance of telomere length
- This leads to cellular immortality and tumor formation

Front 129

Organization in the interphase nucleus is "dynamic" and "non-random", how is this organization maintained?

Back 129

Nuclear matrix

Front 130

What is the perceived purpose of the nuclear matrix?

Back 130

To organize the genome into domains that regulate gene expression and cell replication

Front 131

What are the three components of the nuclear matrix?

Back 131

1. Nuclear envelope / Pore Complex / Lamina
2. Nucleolus
3. Internal Nuclear Matrix

Front 132

What would be the consequences if the structure of the interphase nucleus was not organized?

Back 132

- It would be very difficult to maintain normal replication and transcription of chromatin
- This could lead to carcinogenesis and disease

Front 133

Describe the nuclear envelope component of the nuclear matrix.

Back 133

- Double membrane
- Encloses perinuclear space
- Continuous with endoplasmic reticulum (ER)
- Divided into segments, limited by nuclear pores
- Outer membrane faces cytoplasm and contains ribosomes
- Inner membrane contains integral proteins that bind nuclear lamina, which attaches to marginal heterochromatin

Front 134

Describe the nuclear pore complex found in the nuclear matrix?

Back 134

- Lie between segments of nuclear envelope
- ~10nm diameter holes
- Regulates passage of proteins into, and proteins and RNAs out of, the nucleus
- Pores have three strata arranged as octamer
- Proteins in strata termed nucleoporins which act as docking sites for proteins that contain nuclear localization signals (NLSs)

Front 135

How big of proteins can get through the nuclear pore complexes of the nuclear envelope?

Back 135

Proteins smaller than 40kD diffuse through pores; larger proteins are under complex regulation (require nuclear localization signals, NLSs, to get in)

Front 136

What is the function of nucleoporins?

Back 136

- Proteins found on the strata of the nuclear pores
- Nucleoporins in cytoplasmic stratum serve as "docking sites" for proteins with nuclear localization signals (NLSs)

Front 137

What are nuclear localization sequences (NLSs) required for? What common residues do they contain?

Back 137

- NLSs required for import into the nucleus
- Contain positively charged basic amino acids (lysine, K, and arginine, R)
- Must be on surface of protein

Front 138

What do proteins containing NLS sequences bind to? What is an example?

Back 138

- NLS-Receptor Proteins
- Found in cytoplasm
- Ex: importin - heterodimer with two subunits (α and β)

Front 139

Explain how the NLS receptor protein, Importin, transports proteins into the nucleus?

Back 139

- Importin-α binds NLS of nuclear protein
- Complex migrates to pore where importin-β binds a nucleoporin in pore complex
- Energy-dependent transport is used to get through the pore (Ran-GTP)
- Once inside, nuclear protein is released and importin is cycled back through pore

Front 140

How does phosphorylation status affect nuclear import?

Back 140

- Phosphorylation status either enables or inhibits nuclear import
- Some proteins may have to be de-phosphorylated
- Some have to be released by a cytoplasmic masking protein to be recognized by importin; but masking protein requires phosphorylation to release protein

Front 141

What is the purpose of Nuclear Export Sequences (NESs)? What amino acid is common in NES sequences?

Back 141

- Used to export proteins and RNA sequences from the nucleus
- These sequences are leucine-rich

Front 142

Are nuclear pores specific for transport of proteins or RNA?

Back 142

- No, protein and RNA transport are not pore-specific
- Same pore can accommodate both

Front 143

What is the energy source for nuclear import and export?

Back 143

- RanGTP: GTP binding protein
- High concentration of GTP in nucleus, low conc. in cytosol
- Ran-GTP binds to empty nuclear import receptors and cargo-bound export receptors
- They exit via pore to the outside of the cell where GTP is hydrolyzed to GDP

Front 144

Why is it important to understand nuclear import/export ("trafficking")?

Back 144

- Drugs cannot readily penetrate the nucleus
- Nuclear structures are poor targets for therapeutic intervention
- Using knowledge explaining how substances are targeted to nucleus, approaches may be designed that enable development of drugs that can target nuclear structures and genes

Front 145

Where is the nuclear lamina located?

Back 145

- Adjacent to inner nuclear envelope
- Marginal heterochromatin are attached on inside of nucleus
- It is broken up by the pore complexes

Front 146

What are the lamina's proteins called? What is their function?

Back 146

- Lamins A, B, and C
- Lamins A and C interact with the marginal heterochromatin
- Lamin B binds the inner nuclear membrane via the Lamin-B receptor (LBR), an integral membrane protein

Front 147

When the nuclear envelope falls apart, what happens to the Lamin proteins?

Back 147

- Lamin B remains bound to the LBR of the inner nuclear envelope
- This mediates the reformation of the nuclear envelope segments at the start of the next interphase (Lamins A and C attach to Lamin B)

Front 148

What are "Laminopathies"?

Back 148

- Mutations of the LMNA gene (gene that codes for Lamin A and Lamin C)
- Lamins A and C are important for binding to heterochromatin to organize the DNA in the nucleus
- Consequences range from several rare cardiac and skeletal muscular dystrophies (wasting away) to a severe form of premature aging (progeria)

Front 149

What occurs in the Nucleolus?

Back 149

Ribosomal RNA (rRNA) production

Front 150

Each nucleolus has how many chromosomes attached to it? How many genes for rRNA are on each chromosome? How many total genes for rRNA are there per nucleus?

Back 150

10 chromosomes
x ~40 rRNA genes
= 400 rRNA genes/nucleus

Front 151

Describe the nucleolus's membrane?

Back 151

Trick, it doesn't have a membrane!

Front 152

Why does transcription of RNA look like a christmas tree?

Back 152

- Cell has a high demand for ribosomes
- Transcription of rRNA genes occurs constantly, via RNA polymerase I
- RNA transcripts attached to the gene are shorter at the 5' end and longer at the 3' end

Front 153

What makes up the majority of the nuclear matrix?

Back 153

The internal nuclear matrix (~98%)

Front 154

What structures make up the internal nuclear matrix?

Back 154

- Chromatin: DNA and histones (H1, H2A, H2B, H3, H4)
- Non-histones (transcription factors, RNA and DNA polymerases, etc)

Front 155

How are chromosomes situated in the interphase nucleus? How was this proven?

Back 155

- Rabl configuration: telomeres stick to nuclear envelop and centromere is stuck to opposite side (not mammalian cells, although they do occupy assigned territories)
- Laser micro-radiation showed that, once marked by laser burns, chromosomes occupy the same site of the interphase nucleus
- Also, DNA fluorescent in situ hybridization (FISH) showed that chromosomes occupy discrete territories in nucleus

Front 156

What is the inactive X chromosome called? Where is it located in the interphase nucleus?

Back 156

- Barr body
- Always located along the inner nuclear envelope

Front 157

How does the activity/movement of chromosomes change?

Back 157

- Chromosomes move around in their territories
- Activity diminishes as cells become heterochromatic and less transcriptionally active during differentiation
- This is reversed when cells become tumorigenic

Front 158

Are nuclear matrix proteins disease-specific and tissue-specific? What is the significance of this?

Back 158

- Yes
- Some nuclear matrix proteins in normal cells and cancer cells are different (useful in diagnosis)

Front 159

What is Tg?

Back 159

- Generation time
- Time it takes for the cell to go through four cell-cycle phases

Front 160

What are the four phases of the cell-cycle?

Back 160

- G1 phase (Gap 1)
- S-phase (synthesis of DNA)
- G2 phase (Gap 2)
- M-phase (mitosis)

Front 161

Which phases make up interphase?

Back 161

- G1 phase (Gap 1)
- S-phase (synthesis of DNA)
- G2 phase (Gap 2)
(NOT MITOSIS)

Front 162

What can a cell do during the G1 phase (Gap 1)?

Back 162

- Senescence (i.e., Go = nothing happens)
- Differentiation (also Go) = specialize and never divide
- Apoptosis = cell death
- Proliferation = entry into cell cycle (prepare for S-phase)

Front 163

What happens to a cell during S-phase?

Back 163

- Synthesis of DNA
- Chromosomes are duplicated from 2N to 4N
- Histones are synthesized to make up the new chromosomes

Front 164

What happens to a cell during G2 phase (Gap 2)?

Back 164

- Preparation for mitosis occurs
- Late in G2 the centrosome (not centromere) is duplicated
- Hyper-phosphorylation of histone and non-histone proteins occurs late in G2

Front 165

What happens to a cell during M-phase?

Back 165

Mitosis:
- Cells become spherical
- Nuclear membrane disintegrates
- Chromatin condense into chromatids (2/chromosome)
- Chromosomes align on equatorial plate, then chromatids segregate into daughter cells

Front 166

Which length of the cell cycle is predictable in terms of time? How long does it last? Which part is variable?

Back 166

- S, G2, and M are relatively constant (12-24 hours)
- G1 can be very brief of very long (as in Go phase)

Front 167

What are the two types of factors that control the cell cycle?

Back 167

- External factors: 1' messengers
- Internal factors: 2' messengers

Front 168

What kind of molecules are 1' messengers, aka external factors?

Back 168

- Secreted molecules and peptides: growth factors, cytokines, hormones
- Interact with cognate receptors that are in cell membrane or cytoplasm

Front 169

What are some examples of external factors (1' messengers) that activate the cell cycle?

Back 169

- FGFs = fibroblast growth factor
- IGFs = insulin growth factor
- Wnts

Front 170

What are some examples of external factors (1' messengers) that inhibit the cell cycle?

Back 170

- TGF-β = transforming growth factor

Front 171

What are the different kinds of internal factors (2' messengers)?

Back 171

- "Early response" genes: c-myc, fos, jun - rapidly respond to growth signals (~15 min.)
- "Delayed response" genes: cdks and cyclins

Front 172

When CDKs and cyclins are combined, what do they form? What is this unit called?

Back 172

- Heterodimers
- Cyclin dependent protein kinases

Front 173

What do kinases do?

Back 173

Phosphorylate target proteins

Front 174

What is the function of the CDK subunit? What are some examples?

Back 174

- Catalytic subunit
- Phosphorylates the target (substrate)
- CDKs 1, 2, and 4

Front 175

How does the content of each CDK change throughout the cell cycle?

Back 175

It remains constant

Front 176

What is the function of the cyclin subunit? What are some examples?

Back 176

- Regulatory subunit
- Regulates activity of kinase heterodimer
- Cyclins D, E, A, and B

Front 177

How does the content of each cyclin change throughout the cell cycle?

Back 177

It increases (hence name - cycles...cyclin)

Front 178

What are proto-oncogenes? Examples?

Back 178

- When it is mutated the protein it encodes becomes pathologically over-active; thus abnormally stimulating the cell-cycle to induce cancer
- Ex: growth factors, growth factor receptors, early response genes, CDKs/cyclins

Front 179

What residues do kinases phosphorylate?

Back 179

Residues with OH groups:
- Serine
- Threonine
- Tyrosine

Front 180

When is regulation by growth factors predominant?

Back 180

- During early G1 phase
- Diminishes by end of G1 before restriction point (R)

Front 181

What molecules regulate discrete steps during interphase?

Back 181

- Intracellular cyclins
- (although cyclins are important throughout)

Front 182

When do each of the four Cyclins (D, E, A, and B) reach their peak concentrations?

Back 182

- Cyclin D - increases near beginning of G1, peaks late G1, stays high through mitosis
- Cyclin E - peaks at end of G1 phase
- Cyclin A - increases beginning of S phase; peaks at beginning of G2 phase (where it abruptly goes down)
- Cyclin B - Increases during G2 but peaks at beginning of mitosis

Front 183

What does p27 have to do with Cyclins D, E, A, and B?

Back 183

- p27 is a cell-cycle inhibitor (directly inhibits CDK-2 and CDK-4)
- It is high during Go phase
- Drops at beginning of G1 phase

Front 184

What molecule(s) is G1 phase activated by?

Back 184

CyclinD/CDK4 heterodimer (first checkpoint)

Front 185

Describe the activation of Cyclin D which is required for initiating G1 phase.

Back 185

Growth factors induce increase in Cyclin D:
- Wnt growth factors induce increase of cytoplasmic β-catenin
- This migrates to nucleus to activate transcription of c-myc gene
- This activates cyclin D gene
- Growth factors also induce intracellular ras (GTPase-binding protein) which activates MAP kinases to effect cyclin D production

Front 186

What happens to Cyclin D in order to initiate G1 phase?

Back 186

- Cyclin D binds CDK4 to form heterodimer
- CyclinD/CDK4 phosphorylates retinoblastoma (Rb) protein forming P~Rb
- P~Rb releases transcription factor protein E2F
- E2F activates genes encoding Cyclin E and Cyclin A (for 2nd checkpoint)

Front 187

Retinoblastoma (Rb) protein controls the cell cycle how?

Back 187

- Rb inhibits cell-cycle
- Phosphorylated Rb (via CyclinD/CDK4) releases inhibition of protein E2F
- E2F is necessary to activate genes for Cyclin E and A

Front 188

What is the S-phase / 2nd checkpoint activated by?

Back 188

- CyclinE/CDK2
- Cyclin A/CDK2

Front 189

How do you make the cell commit to completing the cell cycle?

Back 189

- Get passed the restriction checkpoint (R)
- E2F production increases amounts of Cyclins E and A
- When E increases it forms the heterodimer CyclinE/CDK2 which breeches the restriction checkpoint
- Now cell is committed to completing cycle

Front 190

How do "DNA replication complexes" get activated for synthesis?

Back 190

CyclinA/CDK2 heterodimer phosphorylates DNA replication complexes which are poised at multiple origins of DNA replication, leading to activation of synthesis

Front 191

How does the cell ensure that all of the DNA is replicated as well as preventing the genome from being replicated more than once?

Back 191

- CyclinA/CDK2 phosphorylates DNA replication complexes
- This ensures that each daughter cell gets exactly what it is supposed to

Front 192

How is mitosis (M-phase) activated?

Back 192

- Dephoshorylation of CyclinB/CDK1 heterodimer
- Mediated by phosphatase: cdc25
- After being dephosphorylated, CyclinB/CDK1 dimer enters nucleus to phosphorylate target proteins

Front 193

When the CyclinB/CDK1 dimer is dephosphorylated it enters the nucleus to phosphorylate target proteins; what changes does this cause? (beginning of M-phase)

Back 193

- Nuclear envelope breakdown (NEB)
- Mitotic spindle assembly
- Metaphase arrest

Front 194

How is anaphase activated?

Back 194

Mid-metaphase by Anaphase Promoter Complex (APC)

Front 195

How many cells are there in the human body?

Back 195

10^13 (ten trillion)

Front 196

What is the full sequence of events that leads to activation of the cell-cycle?

Back 196

1. Stimulation of cell proliferation via external growth factors
2. Transcription of Cyclin D
3. Cyclin D protein produced in cytoplasm
4. Binding of Cyclin D to CDK4 = active heterodimer
5. Phosphorylation of Rb protein and release of E2F protein
6. E2F induced transcription of Cyclins E and A
7. Cyclin E and A proteins produced in cytoplasm; bind to CDK2 = active heterodimers --> DNA synthesis
7a. p53 mediated transcription of p21
7b. p21 inhibition of CyclinE/CDK2 (prevent cell from going through process again)
8. Cdc25 induced dephosphorylation of CyclinB/CDK1 heterodimer
- CyclinB/CDK1 phosphorylation of targets, inducing (i) nuclear envelope breakdown (NEB), (ii) spindle assembly, (iii) metaphase arrest

Front 197

Very generally, what is cancer?

Back 197

- Genetic disease
- Loss of cellular differentiation
- Increased proliferation and invasiveness of cells
- Changes in chromosomes: re-arrangement, loss, gain

Front 198

Very generally, what causes cancer?

Back 198

- Inherited mutations and/or environmental "insults" to DNA during aging
- Cells evolve towards malignancy with age (accumulation of mutations)

Front 199

What are the five gene categories that are mutated in cancer?

Back 199

1. Proto-oncogenes
2. Tumor Suppressor Genes
3. Genes that regulate Apoptosis
4. Genes that Induce Cellular Immortality: Telomerase
5. Genes that Repair DNA

Front 200

When proto-oncogenes are mutated, what are they called?

Back 200

Oncogenes

Front 201

What are the consequences of proto-oncogenes being mutated?

Back 201

- Genes are either over-expressed or have intensified activity
- Either way, leads to UP-regulation

Front 202

What are some examples of oncogenes?

Back 202

- ras (GTP binding protein)
- src (tyrosine kinase cell surface receptor)
- cyclin D
- EGFR (epidermal growth factor receptor)
- Myc (transcription factor)

Front 203

What does a normal tumor suppressor gene do? What happens when it is mutated?

Back 203

- Inhibit cell-cycle normally
- When mutated they no long work

Front 204

What are some examples of tumor suppressor genes?

Back 204

- p21: inhibits CDKs 2 and 4
- p53: activates p21
- Rb: binds E2F (to prevent it from inducing cyclins E and A)
- BRCA: usually repairs broken DNA (common in breast cancer)

Front 205

Which tumor suppressor gene is often mutated in breast cancer?

Back 205

BRCA

Front 206

Why is apoptosis essential?

Back 206

- Must be a balance between cell renewal and cell death
- Diseased cells must be removed
- Stimulation of apoptosis causes tumors to regress

Front 207

What is the sequence of apoptosis?

Back 207

- Macrophages release TNF (tumor necrosis factor)
- TNF binds TNFR cell membrane receptor
- Balance between pro-apoptotic and anti-apoptotic factors is established
- Pro-apoptotic signals induce 'leakiness' of outer membrane of mitochondria
- Cytochrome-C escapes mitos into cytoplasm where it activates Caspase
- Caspase is a protease that destroys chromatin
- Blebs appear on cell surface and cell defoliates = Apoptosis

Front 208

What gene normally inhibits apoptosis? When it is mutated, how can this lead to cancer?

Back 208

- bcl-2
- When mutated it is abnormally active
- Too much inhibition of apoptosis occurs
- Leads to tumor formation (ex: in B cell lymphoma)

Front 209

What gene induces cellular mortality?

Back 209

Telomerase

Front 210

What is the "telomere hypothesis"?

Back 210

When telomere reaches a certain length it sends a signal to cell to become senescent

Front 211

What kind of cells have the ability to restore the telomere length after every cell division?

Back 211

- Embryonic stem cells
- Adult sperm and egg cells (germ cells)

Front 212

What happens if telomerase is mutated in somatic cells?

Back 212

- It may become active
- This would render them immortal
- Usually they die after so many cell cycles
- Can result in carcinogenesis

Front 213

What consequences can occur if genes that repair DNA are mutated?

Back 213

- These proteins can be inactivated
- Clones of abnormal cells arise

Front 214

In what cancer has it been seen that mutations to genes that repair DNA was responsible?

Back 214

Colorectal cancer

Front 215

What are three specific approaches to cancer treatment (to avoid wide-ranging side effects of chemo and radiation)?

Back 215

- Target metastasis
- Target angiogenesis
- Target specific, sick molecules

Front 216

How can targeting metastasis help treat cancer specifically?

Back 216

- Specific protease-inhibitor molecules inhibit a protease enzyme's active site
- In cancer, proteases such as metalloproteinases facilitate metastatic spread by enabling "primary" cancer cells to eat through extracellular matrix
- "TIMPS" = tissue inhibitors of metalloproteinases are being studied in clinical trials

Front 217

How can targeting angiogenesis help treat cancer specifically?

Back 217

- Angiogenesis is process of new blood vessels developing
- Tumors must be within 200μm of blood supply
- If angiogenesis is prevented, tumors can be starved to death

Front 218

How can targeting specific, sick molecules help treat cancer specifically?

Back 218

- Small inhibitor RNAs (siRNAs) target and destroy pathogenic RNAs and viruses via RNA inhibition (RNAi)
- This could be used to target molecules that activate the cell-cycle in tumor cells but not in normal cells

Front 219

How does the use of small inhibitory RNAs (siRNAs) work to target specific, sick molecules, in theory?

Back 219

- siRNAs complementary to pathogenic RNA are introduced to cell
- siRNAs bind to enzyme complex termed RISC
- RISC/siRNA complex base-pairs with complementary pathogenic RNA
- RISC cleaves the pathogenic RNA, eliminating it

Front 220

What are some problems with siRNAs as a specific cancer treatment?

Back 220

- Must prevent "off-target effects" on normal molecules (caused by mis-matches)
- Must develop methods to deliver siRNAs to patients' tumors in vivo

Front 221

What occurs during the Go phase of the cell cycle?

Back 221

- Terminally differentiated cells withdraw from cell cycle indefinitely
- A cell returning from Go enters at early G1 phase

Front 222

What occurs during the G1 phase of the cell cycle?

Back 222

- RNA and protein synthesis
- No DNA synthesis
- Restriction point at end: cell that passes this point is committed to pass into S phase

Front 223

What occurs during the S phase of the cell cycle?

Back 223

- DNA synthesis doubles amount of DNA in cell
- RNA and protein also synthesized

Front 224

What occurs during the G2 phase of the cell cycle?

Back 224

- No DNA synthesis
- RNA and protein synthesis continue

Front 225

What occurs during the M phase of the cell cycle?

Back 225

- Mitosis (nuclear division)
- Cytokinesis (cell division)
- Yields two daughter cells

Front 226

What are the four basic properties of DNA replication?

Back 226

1. Semiconservative
2. Synthesized in 5'-->3' direction
3. A 3'-OH primer and a template are required
4. Semi-discontinuous

Front 227

What does semiconservative DNA synthesis mean?

Back 227

- Produce two molecules with both old and new DNA (strand of each)
- Each strand acts as a template for the synthesis of a new DNA molecule

Front 228

The 3'-OH primer required for DNA replication does what to initiate?

Back 228

3'OH performs a nucleophilic attack on α phosphate of incoming dNTP

Front 229

What does it mean for DNA replication to be semi-discontinuous?

Back 229

- In order to replicate in 5'-->3' direction, there will be a leading strand and a lagging strand
- Only short piece of template is exposed on lagging strand
- This generates Okazaki fragments (each is separated by a gap and piece of RNA)

Front 230

Why is a RNA primer used to begin DNA replication on the lagging strand?

Back 230

- DNA synthesis requires a free 3'-OH primer
- On lagging strand must substitute for RNA primer

Front 231

What are the four basic steps of DNA replication?

Back 231

1. Separation of two complementary strands at origin or replication
2. Formation of replication fork (primers and Okazaki fragments)
3. Chain elongation
4. Removal of primers

Front 232

How many origins of replication are on prokaryote DNA? Eukaryotes?

Back 232

- Prokaryotes: one origin per piece of circular DNA
- Eukaryotes: many origins in order to be most efficient

Front 233

What are the three catalytic activities of E. coli DNA polymerase I?

Back 233

- 5'-->3' polymerase
- 5'-->3' exonuclease
- 3'-->5' exonuclease

Front 234

What is the purpose of the 5'-->3' and 3'-->5' exonucleases of E. coli DNA polymerase I?

Back 234

- Cleave phosphodiester bonds
- Proofreading activity: removes incorrectly base paired nucleotides

Front 235

What is the main difference between the enzymatic function of DNA polymerase I and III?

Back 235

- Pol I has 5'-->3' and 3'-->5' exonucleases
- Pol III has no exonuclease activity (no proofreading)

Front 236

What is the proofreading mechanism of DNA Pol I?

Back 236

- Mispaired 3'OH end of growing strand blocks further elongation
- DNA polymerase slides back to position the mispaired base in the 3'-->5' exonuclease active site
- Mispaired nucleotide is removed
- DNA pol slides forwards and resumes polymerization activity

Front 237

Due to the functions of DNA poly I and III, what role does each primarily play?

Back 237

- DNA Pol III = synthesizes DNA
- DNA Pol I = erases primer and fill gaps; repair

Front 238

What does the term processivity mean?

Back 238

- Number of catalytic events before dissociation from template
- e.g., how many nucleotides are added by DNA pol before it falls off?

Front 239

Does DNA Pol III or I have higher processivity?

Back 239

- DNA pol III is very processive: synthesizes >500,000 nucleotides before dissociating
- DNA pol I is not very processive: synthesizes only 3-200 nucleotides (more involved in repair)

Front 240

What is the role of DNA helicase?

Back 240

Unwinds the double helix of DNA

Front 241

What is the role of single-stranded DNA-binding proteins (SSB)?

Back 241

- Stabilizes single-stranded regions
- Prevents premature re-ligation

Front 242

What is the role of Primase?

Back 242

Synthesizes RNA primer for DNA replication on lagging strand

Front 243

What is the role of DNA Ligase?

Back 243

Connects DNA fragments after RNA primers are replaced with DNA nucleotides by DNA Pol I

Front 244

What enzyme is responsible for removing the RNA primers from DNA (during replication)?

Back 244

DNA Polymerase I

Front 245

Describe the steps of DNA replication on the lagging strand.

Back 245

1. RNA primer is elongated by DNA polymerase III until another stretch of RNA is encountered
2. RNA primer is excised by DNA polymerase I, one ribonucleotide at a time
3. Gap is filled by DNA polymerase I
4. Remaining nick is sealed by DNA ligase

Front 246

Why does the telomere get shortened with successive replications?

Back 246

- Primer dependence and 5' to 3' directionality of DNA polymerase results in incomplete daughter strand synthesis at telomere

Front 247

How does telomerase lengthen the telomere?

Back 247

- Telomerase is a reverse transcriptase that carries its own RNA template
- RNA component of telomerase is complementary to telomere DNA repeats

Front 248

At which atom does the purine base attach to the sugar?

Back 248

N9 (nitrogen on 5-membered ring)

Front 249

At which atom does the pyrimidine base attach to the sugar?

Back 249

N1

Front 250

At what carbon does the glycosidic linkage occur on the pentose sugar?

Back 250

C1'

Front 251

At what carbon does the phosphate linkage occur on the pentose sugar?

Back 251

C5'

Front 252

What is the name of the purine base "adenine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 252

- Ribonucleoside = adenosine
- Ribonucleotide = adenylic acid or AMP

Front 253

What is the name of the purine base "guanine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 253

- Ribonucleoside = guanosine
- Ribonucleotide = guanylic acid or GMP

Front 254

What is the name of the purine base "hypoxanthine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 254

- Ribonucleoside = inosine
- Ribonucleotide = inosinic acid or IMP

Front 255

What is the name of the purine base "xanthine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 255

- Ribonucleoside = xanthosine
- Ribonucleotide = xanthylic acid or XMP

Front 256

What is the name of the pyrimidine base "uracil" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 256

- Ribonucleoside = uridine
- Ribonucleotide = uridylic acid or UMP

Front 257

What is the name of the pyrimidine base "cytosine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 257

- Ribonucleoside = cytidine
- Ribonucleotide = cytidylic acid or CMP

Front 258

What is the name of the pyrimidine base "thymine" when it is in a ribonucleoside (w/o phosphate)? ribonucleotide (w/ phosphate)?

Back 258

- Ribonucleoside = thymidine
- Ribonucleotide = thymidylic acid or TMP

Front 259

What is the starting molecule for purine de novo synthesis?

Back 259

PRPP (phospho-ribosyl-pyrophosphate)

Front 260

Does purine de novo synthesis start with a sugar and phosphate or end with it?

Back 260

Starts with it as base

Front 261

Does pyrimidine de novo synthesis start with a sugar and phosphate or end with it?

Back 261

Adds it at end

Front 262

Which molecules are necessary to build the purine rings and in what order?

Back 262

1. PRPP (ribose and sugar)
2. Glutamine (amino group)
3. Glycine (entire residue; attaches at carbonyl)
4. N-Formyl tetrahydrofolate (aldehyde)
5. Glutamine (amino group)
6. CO2
7. Aspartate (amino group)
8. N-Formyl tetrahydrofolate (aldehyde)

Front 263

What are the steps of the synthesis of de novo purine nucleotide synthesis, starting with PRPP?

Back 263

1. Displacement of pyrophosphate by amino group of glutamine
2. Addition of glycine
3. Formylation by folic acid derivative N-Formyl tetrahydrofolate
4. Transfer of another nitrogen from glutamine
5. 5-member ring closure by dehydration
6. Carboxylation
7. Addition of aspartate
8. Elimination of fumarate (leaves amino group of aspartate)
9. Formylation by another N-Formyl tetrahydrofolate
10. 6-member ring closure by dehydration: forms IMP

Front 264

Once the purine nucleotide IMP is created, how is it differentiated into the more common AMP?

Back 264

1. Aspartate is added to IMP; GTP hydrolyzed; form adenylosuccinate
2. Fumarate is removed leaving behind amino group from aspartate; AMP is formed

Front 265

Once the purine nucleotide IMP is created, how is it differentiated into the more common GMP?

Back 265

1. NAD+ accepts electrons resulting in a carbonyl being formed (produces XMP)
2. Glutamine donates an amino group; ATP is hydrolyzed to form GMP

Front 266

How is the starting substrate PRPP produced?

Back 266

- PRPP synthetase uses ATP to convert D-ribose-5-P to PRPP
- Essentially adds pyrophosphate group to C1' of ribose sugar to activate it for ATase activity

Front 267

Why does PRPP have the pyrophosphate group on it?

Back 267

- This activates the ribose for addition of amino group from glutamine
- Occurs via ATase enzyme

Front 268

Which step is the rate-limiting step of the de novo synthesis of purines?

Back 268

- First step w/ ATase
- Substituting amino group from glutamine for pyrophosphate group on PRPP

Front 269

Which molecule is the first product that contains the completed purine ring?

Back 269

IMP (inosinic acid)

Front 270

How are the monophosphate nucleosides (GMP and AMP, etc) converted to diphosphates and triphosphates?

Back 270

- Base-specific monophosphate kinases convert AMP and GMP to ADP and GDP respectively; phosphate donated by ATP (leaving ADP for both)
- NDP converted to NTP via another non-base specific kinase and again ATP donates phosphate

Front 271

Adenylate kinase maintains what equilibrium among ATP, ADP, and AMP?

Back 271

- 100x ATP
- 10x ADP
- 1x AMP

Front 272

What regulation controls the purine de novo pathway?

Back 272

- End product regulation (GMP/GDP/GTP and AMP/ADP/ATP) inhibits PRPP synthetase, ATase and each branch point (IMP --> XMP or adenylosuccinate)
- Stimulation of branch point synthesis by end-product of opposite branch (i.e. GTP stimulates IMP conversion to adneylosuccinate, which forms AMP; ATP stimulates ITP conversion to XMP, which forms GMP)

Front 273

What steps take place in the degradation of AMP?

Back 273

- Dephosphorylation and deamination to form inosine (either one can go first)
- Phosphorolysis (removal of sugar) to form Hypoxanthine
- Oxidation to form Xanthine
- Further oxidation to form Uric Acid

Front 274

In the degradation of AMP, why would it be important to deaminate first (producing IMP)?

Back 274

- In vigorously exercising skeletal muscle more ATP is needed
- Production of phosphorylated catabolite IMP facilitates resynthesis of ATP

Front 275

In the degradation of AMP, why would it be important to dephosphorylate first (producing adenosine)?

Back 275

- In ischemic or anoxic heart (heart attack) adenosine is needed
- Adenosine is a diffusible compound that is a potent coronary vasodilator, which facilitates oxygen delivery to damaged tissues

Front 276

What steps take place in the degradation of GMP?

Back 276

- Dephosphorylation to guanosine
- Phosphorolysis (removal of sugar) to form guanine
- Deaminatioin to form Xanthine
- Oxidation to form Uric Acid

Front 277

What enzymes are responsible for dephosphorylation during the degradation of purine nucleotides?

Back 277

5'-nucleotidases (5'-NT)

Front 278

What enzyme is responsible for phosphorolysis during the degradation of purine nucleotides?

Back 278

- Purine nucleoside phosphorylase (PNP)
- Removes the pentose sugar

Front 279

What enzymes are responsible for deamination during the degradation of purine nucleotides?

Back 279

- AMP deaminase
- Adenosine deaminase (ADA)
- Guanase

Front 280

What enzyme is responsible for oxidation of hypoxanthine and xanthine during the degradation of purine nucleotides? What does this produce?

Back 280

- Xanthine dehydrogenase (XDH)
- Converts hypoxanthine to xanthine
- Xanthine to uric acid

Front 281

What does xanthine dehydrogenase (XDH) require to oxidize the end products of purine degradation to uric acid?

Back 281

NAD+ = electron acceptor

Front 282

If there is a decrease in intracellular oxygen a protease is activated and converts xanthine dehydrogenase (XDH) to what? What are the implications?

Back 282

Xanthine Oxidase (XO)
- O2 acts as electron acceptor and generates H2O2 (free radicals-->damage)

Front 283

What is the term for overproduction or underexcretion of uric acid?

Back 283

Hyperuricemia

Front 284

Which is the least soluble purine base?

Back 284

Uric acid

Front 285

Why is it bad if there is overproduction or underexcretion of uric acid?

Back 285

- Uric acid is the least soluble purine base and exists at, or near, saturating levels
- When it exceeds saturation limits it leads to deposits in the blood, joints, tissues, and urine = Gout

Front 286

What disorder is characterized by hyperuricemia (high levels of uric acid) due to inherited metabolic abnormalities?

Back 286

Primary gout

Front 287

What disorder is characterized by excessive uric acid production due to a coexisting acquired condition?

Back 287

Secondary gout

Front 288

What are the most likely causes of primary gout?

Back 288

- Excess uric acid production (overexpression of PRPP synthetase and a partial or complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT)
- Defect in excretion (80%) - defect in family of renal urate transport proteins

Front 289

What are some examples of causes secondary gout?

Back 289

Individuals who overproduce or under excrete uric acid in association with drug intake or unusual dietary habits

Front 290

What is the number one treatment of both kinds of gout? What does it do?

Back 290

- Allopurinol
- Inhibits rate of uric acid production
- Analog of hypoxanthine
- Converted to alloxanthine by xanthine dehydrogenase (XDH) which in turn inactivates the enzyme

Front 291

What is "Xanthinuria"?

Back 291

- Inherited disorder
- Second most insoluble purine base (after uric acid)
- Precipitates when overproduced
- Most commonly caused by deficiency of xanthine dehydrogenase (XDH)

Front 292

What causes the "bubble boy syndrome"?

Back 292

- AKA: SCID (severe combined immunodeficiency disease)
- Adenosine deaminase deficiency
- Associated with severe combined immunodeficiency involving T-cell and B-cell dysfunction
- Likely due to accumulation of dATP in lymphocytes
- dATP is an effective inhibitor of ribonucleotide reductase and consequently inhibits DNA synthesis and cell division

Front 293

What happens if there is a deficiency of purine nucleoside phosphorylase (PNP)?

Back 293

- Impairment of T-cell function
- Individuals underexcrete uric acid and over excrete PNP substrates (inosine and guanosine)
- Accumulation of dGTP in T-cells inhibits ribonucleotide reductase and consequently inhibits DNA synthesis and cell division

Front 294

Why is the salvage pathway of purine nucleotide synthesis preferred?

Back 294

- De novo synthesis is metabolically expensive
- At least 5 ATP molecules are consumed per purine formed

Front 295

What is the use of PRPP in the salvage pathway of purine synthesis?

Back 295

- PRPP is used to attach ribose-5-P directly to a purine base
- Adenine + PRPP --> AMP + PPi (via adenine phosphoribosyltransferase, APRT)
- Hypoxanthine or Guanine or Xanthine + PRPP --> IMP or GMP or XMP + PPi (via HPRT)

Front 296

What happens if there is an inherited deficiency of APRT (adenosine phosphoribosyltransferase)?

Back 296

- Inability to salvage adenine (to form AMP)
- Instead adenine is catabolized to 2,8-DHA which is very insoluble and cant lead to urinary stones

Front 297

On average, about how much of catabolized purine nucleotides are excreted as uric acid, while how much is salvaged?

Back 297

- 10% excreted as uric acid
- 90% salvaged

Front 298

What is the major method of purine synthesis?

Back 298

Salvage pathway

Front 299

What inherited disorder of purine salvage synthesis affects the production of IMP, GMP, and XMP?

Back 299

- Lesch-Nyhan Syndrome
- HPRT is lacking or is present at very low levels (<1% of normal)
- HPRT locus is on X chromosome making this disease only affect males

Front 300

What are the symptoms of Lesch-Nyhan Syndrome (lack of HPRT enzyme for purine salvage)?

Back 300

- Aggressive behavior
- Self-mutilation
- Mental retardation
- High levels of uric acid which leads to gout
- Increased rates of de novo synthesis (to compensate)

Front 301

What are the treatments for Lesch-Nyhan Syndrome (lack of HPRT enzyme for purine salvage)?

Back 301

- Allopurinol is administered to relieve complications of gout
- No known treatment for neurological problems however

Front 302

What residues/molecules contribute to the make-up of a pyrimidine residue?

Back 302

- Glutamine (amino group)
- Aspartate (C-C=C-N)
- CO2

Front 303

What are the characteristics of the enzyme that is responsible for the first three steps in the de novo synthesis of a pyrimidine base?

Back 303

- Carbamoyl-phosphate synthetase (CPS II) (2) = uses glutamine as source of amino group and adds to phosphate
- Aspartate transcarbamoylase (2) = combines first product with aspartic acid
- Dihydro-orotase dehydrogenase (DHODH) (3) = removes water from second product
- Abbreviated "CAD"

Front 304

What step is rate-limiting in the de novo pyrimidine synthesis?

Back 304

- First step: CPS II
- Adding amino group from glutamine to phosphate from ATP

Front 305

What is the first pyrimidine synthesized by the de novo synthesis?

Back 305

UMP (uridylic acid)

Front 306

How is the pyrimidine nucleotide CTP generated?

Back 306

- UMP is phosphorylated by a specific and non-specific kinase to UTP
- UTP is converted to CTP by enzyme CTP synthetase; glutamine is source of amino group; ATP is used

Front 307

What is the process of pyrimidine degradation and excretion?

Back 307

- Pyrimidine is dephosphorylated to nucleoside derivative
- If Cytidine, must be deanminated to uridine
- Undergoes phosphorolysis (removal of sugar) to generate Uracil (or thymine if starting with dTMP)

Front 308

How does pyrimidine degradation differ from purine degradation?

Back 308

- Pyrimidines use reduction reactions to break down the ring structure into smaller components (uracil and thymine)
- Purines use oxidation reactions

Front 309

What happens to uracil and thymine in pyrimidine nucleotide degradation?

Back 309

- Additional steps are perform to degrade uracil and thymine to amino acids
- They can then be further degraded by amino acid pathways

Front 310

Why is the salvage pathway much more common than the de novo pathway for pyrimidine synthesis?

Back 310

- De novo costs 5 moles of ATP / mol UMP
- Salvage costs 1 mole of ATP / mol UMP

Front 311

What is the most salvageable pyrimidine products?

Back 311

Nucleosides; these can be used to generate nucleotides

Front 312

What are four inherited disorders of pyrimidine nucleotide metabolism?

Back 312

1. Orotic aciduria-UMP synthase deficiency - excessive orotic acid in urine; often megaloblastic anemia
2. Pyrimidine 5'-nucleotidase deficiency - increased erythrocyte pyrimidine ribonucleotides; leads to anemia
3. Dihydropyrimidine dehydrogenase deficiency - elevated uracil and thymine levels; convulsive disorder characterized by microencephaly
4. Dihydropyrimidinase deficiency - elevated dihydropyrimidines; variable neuro symptoms

Front 313

What is the treatment for Orotic aciduria-UMP synthase deficiency (which leads to excessive amounts of orotic acid)?

Back 313

- Lethal unless treated by pyrimidine replacement, i.e. uridine
- Uracil is an ineffective therapy as it can't be converted into uridine (which can be salvaged into other pyrimidine nucleotides)

Front 314

How are ribonucleotides converted to deoxyribonucleotides?

Back 314

- Reduced by ribonucleotide reductase (RR)
- Substrate is any ribonucleoside diphosphate (NDP)

Front 315

What provides the reducing power for ribonucleotide reductase?

Back 315

- NADPH + H+
(note: not NADH)

Front 316

Which molecule can inhibit ribonucleotide reductase?

Back 316

dATP - this ends up preventing DNA synthesis and cell division

Front 317

How can thymidine be synthesized?

Back 317

- First UDP must be converted to dUDP by ribonucleotide reductase
- dUDP is converted to dUMP
- dUMP is converted to dTMP by thymidylate synthase

Front 318

What are four inherited disorders of pyrimidine nucleotide metabolism?

Back 318

1. Orotic aciduria-UMP synthase deficiency - excessive orotic acid in urine; often megaloblastic anemia
2. Pyrimidine 5'-nucleotidase deficiency - increased erythrocyte pyrimidine ribonucleotides; leads to anemia
3. Dihydropyrimidine dehydrogenase deficiency - elevated uracil and thymine levels; convulsive disorder characterized by microencephaly
4. Dihydropyrimidinase deficiency - elevated dihydropyrimidines; variable neuro symptoms

Front 319

What is the treatment for Orotic aciduria-UMP synthase deficiency (which leads to excessive amounts of orotic acid)?

Back 319

- Lethal unless treated by pyrimidine replacement, i.e. uridine
- Uracil is an ineffective therapy as it can't be converted into uridine (which can be salvaged into other pyrimidine nucleotides)

Front 320

How are ribonucleotides converted to deoxyribonucleotides?

Back 320

- Reduced by ribonucleotide reductase (RR)
- Substrate is any ribonucleoside diphosphate (NDP)

Front 321

What provides the reducing power for ribonucleotide reductase?

Back 321

- NADPH + H+
(note: not NADH)

Front 322

Which molecule can inhibit ribonucleotide reductase?

Back 322

dATP - this ends up preventing DNA synthesis and cell division

Front 323

How can thymidine be synthesized?

Back 323

- First UDP must be converted to dUDP by ribonucleotide reductase
- dUDP is converted to dUMP
- dUMP is converted to dTMP by thymidylate synthase

Front 324

What are four inherited disorders of pyrimidine nucleotide metabolism?

Back 324

1. Orotic aciduria-UMP synthase deficiency - excessive orotic acid in urine; often megaloblastic anemia
2. Pyrimidine 5'-nucleotidase deficiency - increased erythrocyte pyrimidine ribonucleotides; leads to anemia
3. Dihydropyrimidine dehydrogenase deficiency - elevated uracil and thymine levels; convulsive disorder characterized by microencephaly
4. Dihydropyrimidinase deficiency - elevated dihydropyrimidines; variable neuro symptoms

Front 325

What is the treatment for Orotic aciduria-UMP synthase deficiency (which leads to excessive amounts of orotic acid)?

Back 325

- Lethal unless treated by pyrimidine replacement, i.e. uridine
- Uracil is an ineffective therapy as it can't be converted into uridine (which can be salvaged into other pyrimidine nucleotides)

Front 326

How are ribonucleotides converted to deoxyribonucleotides?

Back 326

- Reduced by ribonucleotide reductase (RR)
- Substrate is any ribonucleoside diphosphate (NDP)

Front 327

What provides the reducing power for ribonucleotide reductase?

Back 327

- NADPH + H+
(note: not NADH)

Front 328

Which molecule can inhibit ribonucleotide reductase?

Back 328

dATP - this ends up preventing DNA synthesis and cell division

Front 329

How can thymidine be synthesized?

Back 329

- First UDP must be converted to dUDP by ribonucleotide reductase
- dUDP is converted to dUMP
- dUMP is converted to dTMP by thymidylate synthase

Front 330

What is required for RNA transcription?

Back 330

- DNA template
- Ribonucleoside 5'-triphosphates
- NO primer

Front 331

What are the three major types of RNA and what percent do they make up?

Back 331

- mRNA (5%)
- tRNA (~15%)
- rRNA (~80%)
- snRNA (small nuclear RNA)

Front 332

In prokaryotes how many RNA polymerases are needed to synthesize mRNA, tRNA, and rRNA?

Back 332

Only 1

Front 333

What subunit of RNA polymerase allows it to recognize promoter regions?

Back 333

σ subunit

Front 334

What kind of enzyme is RNA polymerase?

Back 334

Zn2+ metalloenzyme

Front 335

What kind of proofreading mechanism does RNA polymerase have?

Back 335

No 3'-->5' proofreading activity

Front 336

What is the consequence of not having 3'-->5' proofreading activity on RNA polymerase?

Back 336

Error rate is much higher than during DNA synthesis (10^4 - 10^5)

Front 337

What are the three components of the "Operon"?

Back 337

- Promoter = site for binding RNA polymerase
- Operator = binding sites for repressor of activator (not in eukaryotes)
- Structural gene(s)

Front 338

What is the function of the lac operon?

Back 338

- Regulating the production of enzymes that allow for the utilization of lactose as a carbon source

Front 339

Can multiple genes be on one operon in eukaryotes?

Back 339

No, there is a new operon for each gene; unlike in prokaryotes where multiple genes can be controlled by a single operon

Front 340

What are the steps of RNA transcription?

Back 340

- Initiation
- Elongation
- Termination
(- Protein-Independent Termination)

Front 341

Which step is paramount in regulating whether a gene is made into a protein?

Back 341

Initiation of Transcription

Front 342

What are the two regions of the promoter in prokaryotes that are important for the polymerase to recognize?

Back 342

-35 region
-10 region

Front 343

When does the "closed complex" form?

Back 343

- When the RNA polymerase σ unit recognizes -35 region of DNA

Front 344

After the RNA polymerase forms a closed complex at -35 region, what happens?

Back 344

It migrates to -10 region; DNA is unwound and forms an open complex

Front 345

What is the key initiation event of transcription?

Back 345

Transition from closed complex to open complex; this commits transcription to happening

Front 346

As RNA polymerase proceeds past -10 region, what happens to the σ subunit that was important for recognizing the regions?

Back 346

It's released

Front 347

What is the purpose of having different σ subunits on RNA polymerase?

Back 347

They recognize different promoters

Front 348

How does the σ subunit affect RNA polymerase?

Back 348

Increases specificity but decreases affinity

Front 349

What is the energy source for transcription elongation?

Back 349

- Cleavage of phosphate bond between ribonucleotide
- Subsequent hydrolysis of pyrophosphate to inorganic phosphate

Front 350

Can more than one RNA polymerase be involved in transcription of one gene at the same time?

Back 350

- Yes
- Multiple RNA polymerase complexes load onto the promoter region in sequential fashion

Front 351

How processive is RNA polymerase?

Back 351

Highly; it can synthesize thousands of nucleotides before falling off

Front 352

When does a transcription bubble form?

Back 352

- During elongation
- Negative superhelicity in double-stranded DNA facilitates melting of two strands of DNA

Front 353

Ahead of the advancing transcription complex, how is the DNA supercoiled?

Back 353

Positively supercoiled

Front 354

Behind of the advancing transcription complex, how is the DNA supercoiled?

Back 354

Negatively supercoiled

Front 355

Do the mechanisms of transcription termination involve proteins?

Back 355

Yes and No; there is protein-dependent and protein-independent mechanisms

Front 356

What structure do protein-dependent and protein-independent transcription termination mechanisms rely upon?

Back 356

Hairpin loop

Front 357

What interaction does the protein-dependent transcription termination depend upon?

Back 357

Interaction of terminator protein Rho (ρ) with RNA polymerase elongation complex as it pauses at a hairpin loop

Front 358

Describe the Rho (ρ) protein that is important for protein-dependent transcription termination.

Back 358

- Binds to single-stranded RNA
- ATP-dependent (ATPase activity allows it to move towards complex)
- RNA-DNA helicase: disrupts RNA-DNA hybrid, leading to disassembly of elongation complex

Front 359

How is the Rho (ρ) protein able to disrupt transcription and ultimately terminate it?

Back 359

Transcription complex stalls at hairpin loop which signals termination

Front 360

How does protein-independent transcription termination occur?

Back 360

- Hairpin loop (GC rich) causes transcription complex to pause
- Run of adenylates in template bound to run of uridylates in transcript are weakly bound
- Stalling causes RNA-DNA hybrid of A-U pairs to destabilize and disengage the complex
- dA-rU is weak compared to dA-dT (therefore DNA duplex reforms, removing RNA transcript)
- RNA polymerase has less affinity for double-stranded DNA than single-stranded RNA therefore it dissociates

Front 361

How does eukaryotic transcription termination occur?

Back 361

- More similar to protein independent mechanism (GC hairpin stalls transcription complex; AU region of bonding between transcript and template falls apart)

Front 362

How many RNA polymerases are there? What do they synthesize?

Back 362

- I = 18S, 5.8S, and 28S rRNA
- II = mRNA precursors and snRNA
- tRNA and 5S RNA

Front 363

How can you distinguish between the three eukaryotic RNA polymerases besides based on their cellular transcripts?

Back 363

Sensitivity to α-Amanitin (mushroom toxin)
- I = insensitive
- II = strongly inhibited
- III = inhibited by strong concentrations

Front 364

What are some drug inhibitors of transcription?

Back 364

- Rifampicin (inhibits bacterial RNAPs)
- α-amanatin (inhibits euk RNAP II)
- Actinomycin D (intercalates between two G-C base pairs in DNA)
- Daunorubicin (intercalates between base pairs)
- Cordycepin (chain terminator that lacks 3'OH)

Front 365

When are transcription and translation separated in space and time?

Back 365

In eukaryotes (NOT in prokaryotes)

Front 366

How are transcription and translation separated in time and space in eukaryotes?

Back 366

- Nuclear membrane
- Transcription inside nucleus
- Translation outside of nucleus

Front 367

What is a benefit of eukaryotes separating transcription and translation physically?

Back 367

Allows for transcriptional regulation because translation does not begin immediately

Front 368

What are some methods of transcriptional processing in eukaryotes?

Back 368

- Capping to 5' end
- Poly-adenylation (at 3' end)
- Intron splicing by splicosomes

Front 369

What is the purpose of adding a cap to the 5' end of transcripts?

Back 369

- Enhances stability: protection from nucleases / degradation
- Enhances translation efficiency

Front 370

What are examples of 5' capping of transcripts?

Back 370

- 7-methyl-guanosine (added by guanylyltransferase)
- Methyl groups added to riboses (methyl transferases using S-Adenosyl Methionine, SAM, as donor)

Front 371

What kind of RNA can be capped?

Back 371

Only mRNA

Front 372

What is the purpose of adding the poly-A tail to transcripts?

Back 372

- Increases mRNA stability
- Helps in translation

Front 373

What is the mechanism of mRNA splicing in eukaryotes?

Back 373

1. 2'OH attacks 5' splice
2. Newly formed 3'OH attacks 3' splice and exons joined
3. Lariat formed of intron with 2'-5' bond formed (one end is bonded to the middle of the chain, like the letter 'd')

Front 374

What molecules are involved in the precise recognition of splice sites and splicing mechanism?

Back 374

snRNAs (small nuclear RNAs)

Front 375

What plays a key role in alignment and catalysis of splicing introns?

Back 375

RNA molecules (snRNAs)

Front 376

Does splicing require energy?

Back 376

Yes, ATP is involved in unwinding RNA duplex intermediates and catalyzing release of RNPs mRNA precursors and products

Front 377

What is the purpose of introns?

Back 377

Provides another method of generating protein diversity

Front 378

Approximately what percent of human genetic diseases are possibly due to splicing defects?

Back 378

~15%

Front 379

What is MARFAN Syndrome (MRS)?

Back 379

- Defect in fibrillin component of connective tissue
- Splicing defect of fibrillin mRNA
- Leads to defects in microfibrils and elastic fibers surrounding aorta

Front 380

What kind of symptoms does someone with MARFAN Syndrome (MRS) show?

Back 380

- Chest pain
- Dyspnea (difficult/labored breathing)
- MRI shows aorta weakness (and blood seepage) - aortic aneurysm (weak and bulging area in aorta)
- Limbs are disproportionally long and thin, muscle development can be poor, spine can be curved abnormally
- Dural ecstasia - brain and spinal cord surrounded by fluid contained by dura mater

Front 381

What is a possible therapy for MARFAN Syndrome (MRS)?

Back 381

- Blocking TGF-β with neutralizing antibodies
- Leads to normalized lung development

Front 382

Which enzyme hydrolyzes lactose to galactose and glucose?

Back 382

β-galactosidase

Front 383

How are functionally related genes in prokaryotes organized?

Back 383

Into Operons

Front 384

At what level is regulation of operons in prokaryotes primarily exerted?

Back 384

Transcription

Front 385

What is the function of "inducers"?

Back 385

Inducers bind to the repressor to weaken affinity for the operator (thus up-regulating transcription)

Front 386

What are the inducers for the lac operon?

Back 386

- Allolactose
- IPTG

Front 387

In what configuration does the Lac repressor bind to the operator?

Back 387

Tetramer in a tethered dimer configuration

Front 388

What characterizes the binding site of the Lac repressor?

Back 388

Palindromic sequence (symmetrical)

Front 389

What is necessary of glucose concentration before the Lac Operon will be activated?

Back 389

Glucose needs to be used up

Front 390

What happens when glucose is consumed in E.coli?

Back 390

- cAMP levels increase
- cAMP binds to CAP (catabolite activator protein)
- cAMP-CAP binds to promoter, binding facilitates RNAP binding to promoter

Front 391

What is the "glucose effect"?

Back 391

- When glucose levels are high, the lac operon is not activated
- When glucose levels are low, the lac operon is activated so as to create β-galactosidase so that lactose can be used as an energy source

Front 392

Under what circumstances is the Lac Operon ON?

Back 392

- Low glucose
- High lactose

Front 393

Why is the Lac operon off when both glucose and lactose levels are high?

Back 393

- Glucose high, so cAMP levels are low
- cAMP does not bind to CAP
- cAMP-CAP complex necessary to bind to promoter

Front 394

If lactose levels are low, why is the Lac Operon OFF?

Back 394

- Lac Repressor is bound

Front 395

What are four types of molecules that are involved at the promoter for regulation of eukaryotic gene transcription?

Back 395

- Basal Transcription Machinery
- Regulating proteins
- Coactivators
- Insulators

Front 396

What is the function of basal transcription machinery / basal factors?

Back 396

- In response to injunctions from activators, these factors position RNA polymerase at start of protein-coding region
- Composed of RNA Polymerase and general transcription factors (including TATA binding protein)

Front 397

What binds to the core promoter for transcription?

Back 397

Basal Factors / Basal Transcription Machinery

Front 398

What are the two types of regulating proteins for gene regulation in eukaryotes?

Back 398

- Activators and Repressors

Front 399

What do activators and repressors bind to?

Back 399

Enhancer sequences or silence sequences (a distance from the core promoter)

Front 400

What links the basal transcriptional machinery to the activators?

Back 400

Coactivators (mediators)

Front 401

What is the function of insulators?

Back 401

Define regulatory regions of DNA

Front 402

What is the order of assembly of the basal transcription factors?

Back 402

1. TATA-binding protein (TBP) binds first
2. Other basal factors bind at promoter region
3. RNA Polymerase II is recruited to the promoter in a non-phosphorylated state by the basal factors
4. Following assembly into the transcription complex, the C-terminal domain (CTD) of RNA polymerase II is phosphorylated and begins transcriptional elongation

Front 403

What is the rate limiting step of transcriptional initiation?

Back 403

Formation of PIC (pre-initation complex)

Front 404

What are the four structural motifs that are found in DNA-binding proteins in eukaryotes?

Back 404

1. Helix-turn-helix proteins
2. Zinc finger proteins
3. Leucine zipper proteins
4. Helix-loop-helix proteins

Front 405

Describe the structural features of a helix-turn-helix protein.

Back 405

- Two α-helices separated by a β-turn "Recognition helix" fitting in major groove of DNA

Front 406

Describe the structural features of a Zinc finger protein.

Back 406

- Contains zinc bound to Cys and His side chains
- Mediates DNA binding and can also serve as a protein:protein interface

Front 407

Describe the structural features of a leucine zipper protein.

Back 407

- Two α-helices, one with basic residues for DNA binding, one with regularly space Leu for dimerization
- Protein:protein interaction that allows dimerization of transcription factors that contain basic DNA interaction regions (doesn't actually bind DNA)

Front 408

Describe the structural features of a helix-loop-helix protein.

Back 408

- DNA-binding α-helix and two dimerization helices separated by a nonhelical loop
- One helix is smaller, which because of flexibility of loop allows dimerization by folding against another helix
- Larger helix contains DNA-binding regions

Front 409

Which structural motif found in DNA binding proteins in eukaryotes mediates DNA binding of regulatory proteins?

Back 409

Helix-Turn-Helix Motif

Front 410

What are the components required for Protein synthesis?

Back 410

- Amino acids
- tRNAs
- mRNAs (template)
- aa-tRNA synthetases
- Ribosomes
- Initiation, elongation, and termination factors
- ATP and GTP (energy sources)

Front 411

What makes up a ribosome?

Back 411

- rRNA
- protein

Front 412

What are the rRNA and subunits of a prokaryotic ribosome?

Back 412

- rRNA: 23S, 5S = 50S subunit
- rRNA: 16S = 30S subunit
- total: 70S subunit

Front 413

What are the rRNA and subunits of a eukaryotic ribosome?

Back 413

- rRNA: 28S, 5.8S, 5S = 60S subunit
- rRNA: 18S = 50S subunit
- total: 80S subunit

Front 414

What are the properties of the genetic code that gives triplets (called codons)?

Back 414

- Specific (codon always codes for same aa)
- Universal (although mito is slightly different)
- Redundant (degenerate, a given aa may have more than one codon)
- Non-overlapping and comma-less (read from fixed starting point as a continuous sequence)

Front 415

What are the termination codons?

Back 415

UAG
UGA
UAA

Front 416

What is a silent mutation?

Back 416

Codon base is changed to codon that codes for same aa

Front 417

What is a missense mutation?

Back 417

Codon base is changed to codon that codes for different aa

Front 418

What is a nonsense mutation?

Back 418

Codon base is changed to become a termination codon (UAG, UGA, UAA)

Front 419

What is a frameshift mutation?

Back 419

When the reading frame is altered

Front 420

What is the Wobble Hypothesis?

Back 420

tRNAs can recognize more than one codon for a specific amino acid due to 'wobble' at 5' end of tRNA anticodon, forming non-traditional base pairs

Front 421

What are the useful consequences of the Wobble Hypothesis?

Back 421

Don't need 61 tRNAs to read 61 codons

Front 422

What are nonsense suppressors?

Back 422

tRNAs whose anticodons have been mutated such that they incorporate an amino acid at termination codons

Front 423

What are the major steps of protein synthesis?

Back 423

1. Activation of amino acids
2. Initiation
3. Elongation
4. Termination and ribosome recycling
5. Folding and posttranslational processing

Front 424

What occurs during the first step of protein synthesis?

Back 424

- Activation of amino acids by aa tRNA synthetase
- Requires Mg2+, ATP, amino acid, and tRNA
- Creates aminoacyl-tRNA

Front 425

What does it mean to be "polycistronic" vs "monocistronic"? Which kind of cells have which kind?

Back 425

- Polycistronic - mutliple genes / mRNA = prokaryotes
- Monocistronic - single gene / mRNA = eukaryotes

Front 426

How does the ribosome recognize the correct translation initiation codon?

Back 426

- Consensus sequence (Shine-Dalgarno sequence) just upstream from initiation codon
- Pairs with 16S rRNA to position ribosome

Front 427

What transcription factor facilitates initiation? What recognizes the initiation codon?

Back 427

- IF-2 in E.coli and eIF2 in humans
- Initiator tRNA carries N-formyl methionine (bacteria and mitos)
- No formylation in humans

Front 428

What occurs during initiation of protein translation (step 2)?

Back 428

- IF3 keeps 30S and 50S subunits apart
- 30S initiation complex binds
- tRNA carrying N-formyl methionine binds at P site
- 50S subunit binds and GTP is hydrolyzed (70S initiation complex formed)
- Release of IFs

Front 429

What occurs during elongation of protein translation (step 3)?

Back 429

- Elongation factor Tu (EF-Tu) delivers next aminoacyl-tRNA to A site
- Hydrolysis of EF-Tu (this sets rate of synthesis)
- Peptide bond formation by 23SrRNA ribozyme
- Transfer of amino acid in P site to A site
- Deacylated tRNA leaves via E site
- tRNA with amino acids on it moves to P site via EF-G translocase
- Repeat

Front 430

How does protein translation end (step 4)?

Back 430

- Release factor (RF) recognizes stop codon at A site
- RFs change specificity of peptidyl transferase to recognize water instead of amino group as acceptor of activated peptide moiety
- Peptide released

Front 431

What is the initiator tRNA in prokaryotes? In eukaryotes?

Back 431

- Prokaryotes - N-formylmethionine
- Eukaryotes - MET-tRNAi

Front 432

Is the Shine-Dalgarno sequence important for prokaryotes or eukaryotes?

Back 432

Prokaryotes - signals initiation of translation

Front 433

Eukaryotes do not use the Shine-Dalgarno sequence to indicate where translation should begin; how do they know where to begin?

Back 433

- In eukaryotes the AUG nearest the 5' end of the mRNA is generally utilized as the initator MET
- 40S ribosome loads at the 5' end of the mRNA and "scans"
- Stepwise movement requires ATP and helicases

Front 434

What translational initiation factor in eukaryotes recognizes and binds the 5' cap of mRNA and promotes binding of mRNA to 40S subunit?

Back 434

eIF4E (subunit of eIF4)

Front 435

What does the Polio Virus have to do with the eukaryotic translation initiation factor eIF4E?

Back 435

Causes proteolysis of cap binding protein allowing its own uncapped mRNA to be translated

Front 436

What does Diptheria Toxin do?

Back 436

- Blocks eukaryotic translation by inhibiting translocation
- ADP ribosylation of EF2-translocase

Front 437

What are the different types of lipids?

Back 437

- Fatty acids
- Waxes
- Sterols
- Phospholipids
- Fat-soluble vitamins
- Mono-, di-, and tri-glycerides

Front 438

What are the main biological functions of lipids?

Back 438

- Energy storage
- Signaling molecules

Front 439

What are the major structural components of biological membranes?

Back 439

** Glycerophospholipids (phospholipids)
- Also sphingomyelin and cholesterol

Front 440

What is the structure of phospholipids?

Back 440

- Glycerol backbone
- 2 ester linkages to fatty acid-derived tails
- C1 = C16 and C18 saturated FA
- C2 = C18 and C20 unsaturated FA
- Phosphodiester linkage of polar head group to C3

Front 441

What molecule is a common precursor in the synthesis of phospholipids and triglycerides

Back 441

Phosphatidic Acid

Front 442

Where is phosphatidic acid (PA) synthesized (de novo)?

Back 442

- Endoplasmic Reticulum (ER)
- Outer mitochondrial membrane

Front 443

How is phosphatidic acid (PA) synthesized (de novo)?

Back 443

- Glucose derivative dihydroxyacetone phosphate is reduced to Glycerol-3-phosphate (alternatively, glycerol can be phosphorylated to glycerol-3-P)
- 2 FA (from acyl-CoA derivatives) are added to glycerol-3-P by acyl transferases to create Phosphatidic Acid (PA)

Front 444

What molecule is very important in the synthesis of phosphatidic acid (PA)?

Back 444

Dihydroxyacetone Phosphate (glycolytic intermediate)

Front 445

Alternatively, how can DAG be used to synthesize Phosphatidic Acid (PA)?

Back 445

Diacylglycerol (DAG) can be phosphorylated by DAG kinase to PA

Front 446

What are phosphatidates? Examples?

Back 446

- Derivatives of phosphatidic acid (PA)
- Additional polar group is esterified to phosphate moiety
- Examples: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI)

Front 447

What is necessary for phosphatidic acid (PA) to be converted to the various phosphatidates derivatives?

Back 447

- Hydroxyl group must be activated by attachment of cytidine diphosphate (CDP) nucleotide
- Cytidine monophosphate (CMP) is displaced in a nucleophilic attack by other hydroxyl

Front 448

With what pathway does the phosphatidic acid get activated by CDP in order to form phosphatidate derivatives?

Back 448

Kennedy Pathway (two ways)
1. 1,2-diacylglycerol is activated with CDP (polar head group remains inactive)
2. CDP-activated polar head group attaches to phosphate of phosphatidic acid

Front 449

Eukaryotic cells can form phosphatidates from PA by one or two strategies? What pathway is this?

Back 449

Kennedy Pathway
- Both strategies:
1) DAG activated w/ CDP
2) CDP-activated polar head group attached to PA

Front 450

Prokaryotic cells can form phosphatidates from PA by one or two strategies? What pathway is this?

Back 450

Kennedy Pathway
- Only one strategy:
1) DAG activated w/ CDP

Front 451

What are the important functions of CTP?

Back 451

1. Nucleic acid biosynthesis
2. Phospholipid biosynthesis (important for activation in formation of phosphatidates)

Front 452

What are the important functions of CTP?

Back 452

1. Nucleic acid biosynthesis
2. Phospholipid biosynthesis (important for activation in formation of phosphatidates)

Front 453

What are the important functions of CTP?

Back 453

1. Nucleic acid biosynthesis
2. Phospholipid biosynthesis (important for activation in formation of phosphatidates)

Front 454

What are the important functions of CTP?

Back 454

1. Nucleic acid biosynthesis
2. Phospholipid biosynthesis (important for activation in formation of phosphatidates)

Front 455

How is phosphatidylglycerol (PG) synthesized?

Back 455

- Phosphatidic Acid (PA) is condensed w/ CTP to form CDP-diacylglycerol (releasing PPi)
- Nucleophilic attack by C1-OH of glycerol 3-P (releasing CMP) to form phosphatidylglycerol 3-P
- Phosphatase cleavage of Pi to leave Phosphatidyl Glycerol (PG)

Front 456

Phosphatidylglycerol (PG) is abundant in bacteria and is used for what?

Back 456

Necessary for protein translocation across membranes

Front 457

How is phosphatidylglycerol (PG) in animals important?

Back 457

- 2nd most abundant phospholipid in lung surfactant
- May be responsible for spreading of surfactant over alveolar wall surfaces of lung

Front 458

Which phospholipid increases during fetal development?

Back 458

Phosphatidylglycerol (PG)

Front 459

How is cardiolipin synthesized?

Back 459

- Phosphatidylglycerol (PG) is condensed with CDP-diacylglycerol to form cardiolipin

Front 460

Where is cardiolipin important in a cell?

Back 460

- Inner mitochondrial membrane (20% of lipid)
- Many enzymes require it for metabolic functions
- Mediates translocation of cholesterol from outer to inner membrane
- Activates mitochondrial cholesterol side chain cleavage
- Imports protein into mitochondrial matrix

Front 461

How is phosphatidylinositol (PI) synthesized?

Back 461

- Condensation of CDP-diacylglycerol with inositol

Front 462

What phosphatidate derivatives is CDP-diacylglycerol important for in the synthesis of?

Back 462

- Cardiolipin
- Phosphatidylinositol

Front 463

What is the primary source of arachidonic acid? What is arachidonic acid required for?

Back 463

- PI (phosphatidylinositol) is primary source of arachidonic acid
- Required for biosynthesis of eicosanoids

Front 464

What is the main function of phosphatidylinositol (PI)?

Back 464

- Functions as negatively-charged building blocks of membranes
- Regulates non-specific electrostatic interactions with proteins

Front 465

What is the function of phosphorylated derivatives of phosphatidylinositol (PI)? What enzyme is responsible for doing this?

Back 465

- These derivatives are involved in regulation of cell structure and metabolism
- Plays a role in signal transduction
- PI kinases convert PI to derivatives

Front 466

What is created when the PI derivative PIP2 (phosphatidylinositol 4,5-bisphosphate) is cleaved?

Back 466

- DAG (diacylglycerol)
- inositol 1,4,5-triphosphate (IP3)

Front 467

What is the response to the cleavage of PIP2 to DAG and IP3 (which is responsible for this signal response)?

Back 467

- Calcium release
- Activation of Protein Kinase C
- IP3 is responsible

Front 468

What is the second most abundant lipid in the membrane phospholipid?

Back 468

Phosphatidylethanolamine (PE)

Front 469

By which Kennedy Pathway strategy is phosphatidylethanolamine (PE) synthesized?

Back 469

- Strategy 2
- Phosphorylation and activation of head group by CMP is followed by condensation with DAG
- Also PS can be decarboxylated to PE

Front 470

Where does synthesis of Phosphatidylethanolamine (PE) take place?

Back 470

- ER
- Also in outer leaflet of inner mitochondrial membrane (by decarboxylation of PS)

Front 471

What is a possible function of Phosphatidylethanolamine (PE)?

Back 471

Membrane fusion/fission events

Front 472

Which neurotransmitter is Phosphatidylethanolamine (PE) a precursor of?

Back 472

N-acylethanolamine (in the brain)

Front 473

Phosphatidylethanolamine (PE) can donate its ethanolamine group towards the synthesis of what?

Back 473

- Glycosylphosphatidylinositol anchors (GPI) for many cell-surface proteins
- Plasmalogens

Front 474

How is phosphatidylserine (PS) synthesized?

Back 474

Derived from PE or PC via one of two head-group exchange reactions carried out in ER

Front 475

What is phosphatidylserine (PS) necessary for?

Back 475

- Activation of signaling proteins including Protein Kinase C and neutral sphingomyelinase
- Activity of dynamin-1, Na+/K+ ATPases
- Interaction w/ Hsp70 induces formation of ion channels in plasma membrane
- On activated platelets to initiate blood-clotting cascade
- On sperm cells to initiate sperm maturation

Front 476

What happens to phosphatidylserine (PS) during apoptosis?

Back 476

- It is normally enriched in the inner leaflet of membrane bilayer
- Becomes exposed to outside during apoptosis

Front 477

How is phosphatidylcholine (PC) synthesized?

Back 477

- Strategy 2 of Kennedy Pathway: Phosphorylation (phosphocholine) and activation of choline (CDP-choline) is followed by condensation with DAG
- PEMT pathway: methylation of PE (3x)

Front 478

How can phosphatidylcholine (PC) be synthesized from phosphatidylethanolamine (PE)? Compare structures.

Back 478

- PEMT pathway: methylation of PE 3x
- PE head group: -CH2-CH2-NH3+
- PC head group: -CH2-CH2-N(CH3)+
- So H's on PE are substituted for methyl groups

Front 479

For each PC synthesized by the PEMT pathway, what else is produced? What is the significance of this molecule?

Back 479

- 3 S-adenosylhomocysteine molecules
- Independent risk factor for cardiovascular disease

Front 480

Why is there a need for PC to be synthesized by two pathways (Kennedy strategy 2 and PEMT)?

Back 480

- In PEMT- mice, the Kennedy pathway was increased by 50%
- If these PEMT- mice are fed a choline-deficient diet, mice exhibit liver failure in 3 days (choline is necessary for Kennedy pathway)
- Decreased PC in membranes is replaced by PE, which compromises the integrity of the membrane (liver leaks hepatic enzymes leading to inflammation and rapid liver failure)

Front 481

What happens to a wild-type mouse if it is fed a choline deficient diet? Why?

Back 481

- It is normal
- It has the PEMT pathway to generate PC

Front 482

What is the significance of a lack of Phosphatidylglycerol (PG)?

Back 482

Major surfactant deficiency in premature infants relates to lack of PG, even though it comprises less than 5% of pulmonary surfactant phospholipids

Front 483

What is the significance of a lack of cardiolipin synthesis?

Back 483

- Barth syndrome
- Causes infantile death
- Mutation in gene coding tafazzin (enzyme involved in synthesis of cardiolipin)
- Cardiolipin is important for ATP production; w/o enough you get abnormal mitos
- Cardiomyopathy and general weakness
- Supplemental cardiolipin treats symptoms and prevents infections

Front 484

If the asymmetrical transbilayer distribution of PE in sarcolemmal membranes in the heart is altered, what can happen? What causes this dis-regulation?

Back 484

- Leads to sarcolemmal disruption and irreversible cell damage
- Caused by ischemia

Front 485

What alternative linkage can there be in a phospholipid at the C1 of glycerol? How is this made?

Back 485

- Ether linkage (as opposed to an ester linkage)
- Synthesized by dihydroxyacetone, long chain fatty acids and long-chain fatty alcohols (ether linkage)

Front 486

Where is there a higher level of ether-linked lipids (as opposed to the usual ester-linked lipids)?

Back 486

- Vertebrate heart tissue
- Plasma membrane of metastatic cancer cells

Front 487

What is the term for "phosphatidates" that have ether linkages at C1 instead of ester linkages? How else do they differ?

Back 487

Plasmalogens
- Ether linked fatty acyl chain at C1 is unsaturated

Front 488

Which type of enzyme is used to catalyze the desaturation reaction for converting the saturated C1 fatty acyl chain to an unsaturated chain?

Back 488

Mixed-function oxidase

Front 489

Where are plasmalogens commonly found?

Back 489

Heart - half of heart phospholipids are plasmalogens

Front 490

What is usual and unusual about the phospholipid structure of Platelet-Activating Factor (PAF)?

Back 490

- Ether-linked (less normal) fatty acyl chain is usually saturated (normal)
- C2 contains an acetyl residue instead of a long-chain fatty acid (not normal)
- Head group is usually choline

Front 491

What are the consequences of Platelet-Activating Factor (PAF) having an acetyl residue at C2 instead of a long-chain fatty acid?

Back 491

Increases solubility enabling it to function in an aqueous environment

Front 492

What kind of cells make platelet-activating factor (PAF)?

Back 492

- Platelets
- Leukocytes
- Monocytes
- Endothelial cells

Front 493

What are the functions of platelet-activating factor (PAF)?

Back 493

- Mediates allergic and inflammatory responses
- Released from leukocytes (basophils) and induces platelet aggregation and release of serotonin (vasoconstrictor) from platelets
- Stimulates smooth muscle contraction and activation of immune cells
- Mediates hypersensitivity and anaphylactic shock

Front 494

How is Platelet-Aggregating Factor (PAF) involved in allergic responses?

Back 494

- Mediates hypersensitivity and anaphylactic shock - a severe and sometimes fatal immune response
- Causes these effects by binding to an extracellular receptor on target cells

Front 495

What is the head group on phosphatidic acid (PA)?

Back 495

No head group: just an H

Front 496

What is the head group on phosphatidylethanolamine (PE)?

Back 496

Ethanolamine:
-CH2-CH2-NH3+

Front 497

What is the head group on phosphatidylcholine (PC)?

Back 497

Choline:
-CH2-CH2-N(CH3)3+

Front 498

What is the head group on phosphatidylserine (PS)?

Back 498

Serine:
-CH2-CH-(COO-)-NH3+

Front 499

What is the head group on phosphatidylglycerol (PG)?

Back 499

Glycerol:
-CH2-CH(OH)-CH2-OH

Front 500

What is the head group on phosphatidylinositol 4,5-bisphosphate (PIP2)

Back 500

Myo-inositol 4,5-bisphosphate:
- 6 membered ring w/ 3 free OH groups and 2 phosphates (where OH's bonded to P's)

Front 501

What is the head group on cardiolipin?

Back 501

Phosphatidylglycerol

Front 502

What are the two signaling pathways we should know performed by phospholipids?

Back 502

- PLC cleavage, activation of Ca2+ release by IP3 and PKC activation
- Anchoring of membrane-associated proteins (targeting sequences needed to direct them to membrane)

Front 503

Where is PIP2 located and used for?

Back 503

- Found on cytoplasmic side of plasma membrane
- Serves as a specific binding site for a subset of cytoskeletal and soluble proteins
- Also serves as a reservoir of messenger molecules that are released inside cell in response to extracellular signals that interact w/ spec receptors on outside surface of plasma membrane

Front 504

What are the effects of IP3 cleavage from PIP2?

Back 504

- IP3 signals Ca2+ release from ER
- Ca2+ has a variety of intracellular effects, one of which is working with DAG to activate Protein Kinase C (PKC)
- PKC has many intracellular substrates and they perform many functions when phosphorylated

Front 505

What kind of proteins can be covalently attached to the outer surface of the plasma membrane? How are they linked?

Back 505

- Extracellular proteins (e.g., lipoprotein lipase, acetylcholine esterase)
- Glycosyl-phosphatidyl-inositol (GPI) anchor

Front 506

Describe the structure of a GPI anchor.

Back 506

- Phosphatidylinositol derivatives contain a short oligosaccharide (N-acetylglucosamine and mannose) covalently joined to carboxyl-terminal residue of protein through phosphatidylethanolamine

Front 507

How are GPI-linked proteins usually arranged?

Back 507

Arranged in clusters

Front 508

How can GPI-linked proteins be released?

Back 508

Phospholipase cleavage

Front 509

Are intracellular proteins capable of being linked by GPI-linkages?

Back 509

No, GPI-linkages are only for extracellular proteins

Front 510

How are intracellular proteins linked to the membrane?

Back 510

- Long-chain fatty acids (e.g., palmitic acid (16:0) attached to an internal Cys resi; muristic acid (14:0) attached to an N-terminal glycine resi)
- Isoprenoids such as polyisoprene units (farnesyl [C15] and geranylgeranyl [C20]) that attach to carboxyl-terminal Cys resi)

Front 511

How do membrane-associated proteins get linked to the protein?

Back 511

- Targeting sequences in primary translation products direct these proteins to membranes

Front 512

Where are sphingolipids found?

Back 512

- Membranes of all cells
- Highest conc. in cells of central nervous system

Front 513

What is the general structure of a sphingolipid?

Back 513

- Sphingosine backbone: similar to glycerol except:
- C3 has unsaturated 15C chain (not ester linkage)
- C2 has amide linkage (not ester)
- C1 has head group

Front 514

What are the three subclasses of sphingolipids?

Back 514

- Sphingomyelins (phosphocholine or phosphoethanolamine as polar head group)
- Glycosphingolipids (one (cerebroside) or more (globosides) sugars attached to C1
- Gangliosides (polar head groups comprised of oligosaccharides w/ one or more N-acetylneuraminic acid at termini)

Front 515

Where are sphingomyelins highly prevalent?

Back 515

Myelin - membranous sheath surrounding and insulating axon of some neurons

Front 516

Which of the three subclasses of sphingolipids contain a phosphate group?

Back 516

Only sphingomyelins (not glycosphingolipids or gangliosides)

Front 517

What is the base sphingolipid called (with just an H as the head group)?

Back 517

Ceramide

Front 518

What sphingolipid has a clinical relevance to cholera toxin?

Back 518

A specific ganglioside located on the lumenal side of intestinal mucosal cells specifically binds cholera toxin, which stimulates excess secretion that results in severe diarrhea

Front 519

What are the four steps in the biosynthesis of sphingolipids?

Back 519

1. Synthesis of 18C amine sphinganine from palmitoyl CoA and serine; condensation reaction is catalyzed by serine palmitoyltransferase, driven by cleavage of thioester bond of palmitoyl CoA and release of CO2
2. Attachment of FA in amide linkage to yield N-acylsphinganine
3. Desaturation of sphinganine moiety to form N-acylsphingonine (ceramide)
4. Attachment of head group to produce a sphingolipid

Front 520

How are gangliosides synthesized from the basic structure ceramide?

Back 520

- Addition of sugar residues
- Requires activated sugars (including UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and CMP derivative of N-acetylneuraminic acid (CMP-NeuAc))
- Sialic acid residue attached; then additional sugars and/or sialic acid residues can be further added

Front 521

What molecule provides the reducing power for glycerophospholipid and sphingolipid synthesis?

Back 521

NADPH

Front 522

What are the similarities of sphingolipids and glycerophospholipid synthesis?

Back 522

- NADPH provides reducing power
- Fatty acids enter pathway as their activated CoA derivatives

Front 523

How does the head group attachment differ for sphingolipids and glycerophospholipid synthesis?

Back 523

- PC, rather than CDP-choline, serves as the donor of phosphocholine in synthesis of sphingomyelin

Front 524

What kind of linkage connects the head groups of glycolipids (cerebrosides and gangliosides)?

Back 524

Glycosidic linkage

Front 525

Where does degradation of sphingolipids and glycerophospholipids occur?

Back 525

Lysosomes

Front 526

What enzymes specifically cleave phospholipids? What are the four main ones to know?

Back 526

Phospholipases: A1, A2, C, D

Front 527

What is the specific function of phospholipase A1?

Back 527

Removes fatty acids from C1 position of glycerol backbone (permits re-esterification rxns w/ other fatty acyl CoAs)

Front 528

What is the specific function of phospholipase A2?

Back 528

Removes fatty acids from C2 position of glycerol backbone (permits re-esterification rxns w/ other fatty acyl CoAs)

Front 529

What is the specific function of phospholipase C?

Back 529

Cleaves C3 phosphodiester to produce diacylglycerol

Front 530

What is the specific function of phospholipase D?

Back 530

Cleaves C3 phosphodiester to produce phosphatidic acid

Front 531

Degradation of sphingolipids produces what?

Back 531

Sphingosine (backbone) and fatty acids

Front 532

What are the disorders of sphingolipid degradation called?

Back 532

Sphingolipidoses

Front 533

What happens when there is a sphingolipidose?

Back 533

- Accumulation of substrate in lysozyme of tissue responsible for catabolism
- Includes phagocytic cells of reticuloendothelial system, located primarily in the liver, spleen, and bone marrow, and brain

Front 534

Why is the brain particularly vulnerable to sphingolipidose (disorder of sphingolipid degradation)?

Back 534

Brain contains highest concentration of gangliosides; if they aren't catabolized properly they will build up in tissues

Front 535

What are two specific sphingolipidose diseases (abnormal accumulation of sphingolipids)?

Back 535

1. Tay-Sachs Disease
2. Niemann-Pick Disease

Front 536

What is Tay-Sachs disease a disorder of? What are the symptoms?

Back 536

Ganglioside degradation
- Caused by deficiency in β-hexosaminidase
- Manifested in brain
- Neurons become swollen w/ lipid filled lysozymes and infants display motor weakness and retarded psychomotor skills
- Demented and blind by age 2 and usually die by age 3
- Aggravated by high turnover of ganglioside during neonatal period

Front 537

What is Niemann-Pick disease a disorder of? What are the symptoms?

Back 537

- Deficiency in sphingomyelinase (the enzyme that degrades sphingomyelin into ceramide and phosphocholine)
- Results in severe mental retardation but most of lipids are deposited in liver and spleen (enlarging them)
- Death usually in early childhood

Front 538

What kind of lipids act as short-range messengers? What kind of hormones are these?

Back 538

Eicosanoids
- Paracrine hormones

Front 539

What are the precursors of eicosanoids?

Back 539

- Unesterified arachidonic acid [20:4] which is made from:
- Essential FA linoleic acid [18:2]

Front 540

How is the precursor of eicosanoid synthesis obtained?

Back 540

Arachidonic acid from the membrane is cleaved by phospholipase A2 in response to hormonal or other stimuli

Front 541

Do eicosanoids have a short or long half-life? Why?

Back 541

- Short half-life
- Rapid turnover by mitochondrial and peroxisomal β-oxidation pathways

Front 542

What are the three classes of eicosanoids?

Back 542

- Prostaglandins
- Thromboxanes
- Leukotrienes

Front 543

How are prostaglandins and thromboxanes synthesized?

Back 543

- Cyclooxygenase pathway
- Arachidonic acid becomes "cyclized" and "oxidized" by prostaglandin G/H synthase (PGS)
- Occurs in smooth ER

Front 544

What are the functions of the bifunctional enzyme prostaglandin G/H synthase (PGS)?

Back 544

- Important in conversion of arachidonic acid to prostaglandins and thromboxanes
- Has both cyclooxygenase (COX) and glutathione-dependent peroxidase (PG hydroperoxidase) activity

Front 545

The "COX" (cyclooxygenase) function of prostaglandin G/H synthase (PGS) requires what?

Back 545

- Requires 2 molecules of O2 for catalyzing cyclization of C8-C12 of arachidonic acid
- This forms prostaglandin G2 (PGG2)

Front 546

What structural feature distinguishes prostaglandins from the other eicosanoids?

Back 546

5-membered ring (C8-C12)

Front 547

What are the two isoforms of prostaglandin G/H synthase (PGS)?

Back 547

- PGS-1/COX-1 - constitutively expressed in most tissues and cells
- PGS-2/COX-2 - inducible expression at limited number of sites (e.g. inflammation, pain, fever)

Front 548

How are thromboxanes synthesized?

Back 548

- After prostaglandin G2 (PGG2) is created by prostaglandin G/H synthase (PGS)
- It is converted to prostaglandin H2 (PGH2)
- Then it is converted to thromboxane by thromboxane synthase (in platelets)

Front 549

What is the structural change when a prostaglandin is converted to a thromboxane?

Back 549

Cyclopentane ring converted to a 6-membered Oxygen containing (oxane) ring

Front 550

What are the important functions of prostaglandins?

Back 550

- Stimulate uterine smooth muscle contractions during menstruation/labor
- Elevates body temperature and causes inflammation and pain
- Affects blood flow to specific organs and responsiveness of certain tissues to hormones such as epi and glucagon

Front 551

How is prostacyclin (PGI2) synthesized and what is its function?

Back 551

- Change in functional group around cyclopentane ring
- Inhibits platelet aggregation and dilates blood vessels

Front 552

What is the function of the prostaglandin derivative PGE2?

Back 552

Promotes the clotting process

Front 553

What produces thromboxanes? How do they function?

Back 553

- Produced by platelets
- Act in platelet aggregation and formation of blood clots, and reduction of blood flow to site of clot

Front 554

How is prostaglandin synthesis regulated?

Back 554

- PGS-2/COX-2 (prostaglandin G/H synthase (PGS)) is only expressed in certain tissues and cells (activated macrophages and monocytes)
- Activated in response to stimulation by a number of agents including platelet activating factor (PAF)
- Inhibited by glucocorticoids

Front 555

What is PGS-2 responsible for?

Back 555

Elevated levels of prostaglandin, which causes inflammation

Front 556

How do corticosteroids affect prostaglandins? What are the consequences?

Back 556

- They inhibit phospholipase A2 (this decreases production of arachidonic acid, a necessary precursor for prostaglandins)
- Used as anti-inflammatory agents

Front 557

What are NSAIDs used for?

Back 557

- Non-steroidal anti-inflammatory agents
- Block prostaglandin and thromboxane synthesis from arachidonic acid
- Inhibits COX pathway of PGS (both isoforms)

Front 558

What kind of drug is aspirin and how does it specifically function?

Back 558

- NSAID
- Covalently acetylates COX and blocks active site
- Wide-ranging effects: decreased inflammation, fever, pain, and blood clotting

Front 559

What are the side effects of NSAIDs? How?

Back 559

- Stomach irritation and more serious conditions
- Inhibition of COX-1
- NSAIDs that are specific for COX-2 were developed to prevent severe pain but connected with increased risk of heart attack and stroke

Front 560

What are the contradicting effects of prostaglandins and thromboxanes?

Back 560

- Prostacyclin prevents blood clotting
- Thromboxane prevents blood clotting

Front 561

Which of the eicosanoid types is characterized by its linear structure?

Back 561

Leukotrienes

Front 562

How are leukotrienes synthesized? Which synthesizes the precursor to most important leukotrienes?

Back 562

- Lipoxygenase adds one O2 to arachidonic acid
- 5-lipoxygenase (5-LO)

Front 563

What kind of enzyme is lipoxygenase?

Back 563

- Mixed-function oxidase
- Found in heart, brain, lung, and spleen

Front 564

What does 5-lipoxygenase use as a precursor? What does it synthesize? What happens to this molecule?

Back 564

- Converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) via oxygen attack
- 5-HPETE is unstable and is converted by a peroxidase to more stable 5-HETE
- 5-HPETE can also be converted to epoxide called leukotriene A4 (LTA4) via a synthase

Front 565

What is the structurally defining feature of leukotrienes?

Back 565

Four double bonds
(and no 5 or 6-membered ring structures)

Front 566

What are lipoxins?

Back 566

Products of multiple lipoxygenase pathways that are characterized by hydroxyl groups attached to C5 and C6

Front 567

What molecules are "pro-inflammatory"?

Back 567

Most leukotrienes

Front 568

Which molecule appears to inhibit leukotrienes?

Back 568

Lipoxins (anti-inflammatory)

Front 569

What disorders are associated with elevated levels of leukotrienes?

Back 569

- Some inflammatory and hypersensitivity disorders
- E.g., asthma and anaphylactic shock

Front 570

Which molecule regulates 5-lipoxygenase (5-LO)? How?

Back 570

- 5-lipoxygenase-activating protein (FLAP)
- Integral nuclear membrane protein that binds arachidonic acid and facilitates the substrate interaction of this enzyme
- Several inhibitors of leukotriene synthesis bind to FLAP and inhibit its function

Front 571

What is the function of the leukotriene LTB4?

Back 571

- Chemotactic agent
- Attracts neutrophils to combat infection

Front 572

Which molecules induce contraction of smooth muscle lining airways to lungs? When does this happen?

Back 572

Leukotrienes: LTC4, LTD4, LTE4
During anaphylactic shock

Front 573

How do the clinically important drugs in asthma (montelukast/Singulair and zafirlukast/Accolate) help?

Back 573

- Block binding of leukotrienes to lung smooth muscle cell receptor
- Prevents contraction of smooth muscle lining airway to lungs

Front 574

How does the clinically important drug for asthma (Zileuton/Zyflo) help?

Back 574

Blocks 5-LO activity, preventing synthesis of leukotrienes (LTC4, LTD4, LTE4) that induce contraction of smooth muscle lining airway

Front 575

How are polyisoprenoids different from other lipids?

Back 575

They are derived from 5-Carbon isoprene units, rather than fatty acids

Front 576

What are some important polyisoprenoids?

Back 576

- Cholesterol
- Steroid hormones
- Bile acids
- Fat-soluble vitamins (A, D, E, and K)
- Ubiquinone
- Protein-membrane anchors (Farnesyl and Geranylgeranyl)
- Dolichol

Front 577

What is the precursor for steroid hormones and bile acids?

Back 577

Cholesterol

Front 578

What is cholesterol synthesized from?

Back 578

27 molecules of acetate

Front 579

What are the four key steps to cholesterol biosynthesis?

Back 579

1. Condensation of 3 acetate units to form a 6-carbon intermediate: Mevalonate
2. Conversion of mevalonate to activated isoprene units (5C)
3. Polymerization of six 5C isoprene units to form 30C linear squalene
4. Cyclization of squalene to form four rings of steroid nucleus, followed by a further series of changes (oxidations, removal or migration of methyl groups) to produce cholesterol

Front 580

What is the rate-limiting step of cholesterol biosynthesis?

Back 580

- Reduction of HMG-CoA to mevalonate
- 2 molecules of NADPH donate 2 e- each
- Catalyzed by HMG-CoA reductase (major point of regulation for cholesterol synthesis)

Front 581

How are steroids different than cholesterol/sterols?

Back 581

Oxidized sterols (have sterol nucleus, but lack alkyl chain attached to ring D of cholesterol); they are also more polar

Front 582

Which steroid drugs have potent anti-inflammatory activities? How is their function mediated?

Back 582

- Prednisone and prednisolone
- Mediated in part by inhibition of arachidonate release by phospholipase A2
- Consequent inhibition of synthesis of eicosanoids
- Useful for treatment of asthma and rheumatoid arthritis

Front 583

What is the precursor for the lipid-soluble vitamins A, D, E, and K?

Back 583

Cholesterol

Front 584

How is Vitamin D synthesized?

Back 584

- Formed in skin from 7-dehydrocholesterol in a photochemical rxn driven by UV from sunlight

Front 585

What is the function of Vitamin D?

Back 585

- Following enzymatic conversion in liver and kidney to 1,25-dihydroxycholecalciferol, it becomes a hormone that regulates calcium uptake in intestine and calcium levels in kidney and bone

Front 586

What happens if you have a vitamin D deficiency?

Back 586

Defective bone formation

Front 587

How does Vitamin A / retinol function?

Back 587

Functions as a hormone and as visual pigment of eye

Front 588

Which vitamin is important for the treatment of severe acne and wrinkled skin?

Back 588

Vitamin A derivative: retinoic acid (found in tretinoin or Retin-A)

Front 589

What is the function of Retinal (vitamin A derivative)?

Back 589

Pigment that neuronal initiates the response of rod and cone cells of retina to light, producing a neuronal signal to the brain

Front 590

What does a deficiency in Vitamin A cause?

Back 590

- Dryness of skin
- Dryness of eyes and mucous membranes
- Retarded development
- Growth and night blindness

Front 591

What is the function of Vitamin E?

Back 591

Hydrophobic compound associated with membranes, biological anti-oxidant

Front 592

What does a deficiency in Vitamin E cause?

Back 592

Very rare but leads to fragile erythrocytes

Front 593

What is the significance of Vitamin K?

Back 593

- Aromatic ring undergoes a cycle of oxidation and reduction during formation of active prothrombin
- Prothrombin is a blood plasma protein essential in blood clotting

Front 594

What does a deficiency in Vitamin K cause?

Back 594

Very rare but slows blood clotting

Front 595

What kind of molecule is Warfarin? What does it treat?

Back 595

- Isoprenoid
- Inhibits formation of active prothrombin (anticoagulant used to treat excessive blood clotting)

Front 596

What kind of molecule is Ubiquinone or Coenzyme Q? What does it do?

Back 596

- Isoprenoid
- Lipophilic electron carriers in oxidation-reduction reactions
- Drives ATP synthesis in mitochondria

Front 597

What kind of molecule is Dolichol? What does it do?

Back 597

- Isoprenoid
- Intermediate in activation of sugar units destined for attachment to proteins and other lipids

Front 598

What kind of molecules are bile acids and their salts? What do they do?

Back 598

- Isoprenoids
- Polar/hydrophobic cholesterol derivatives that act as detergents in the intestine
- Emulsify dietary fats to make them more readily accessible to digestive lipases
- In liver they aid in digestion

Front 599

What technique can be used to study the structure and fundamental properties of membranes?

Back 599

- Freeze Fracture Technique
- Cells are cracked open at liquid N2 temperature
- Membranes split within hydrophobic interior because free energy of binding is lower in this region
- Proper preparation of membrane reveals the interior of the phospholipid bilayer as a smooth surface and intramembrane particles that reflect the distribution of membrane proteins

Front 600

What is a common protein modification on integral membrane proteins?

Back 600

Oligosaccharide (sugar) chain exposed to exterior side

Front 601

What is the "glycocalyx"?

Back 601

- "Sugar coat"
- Negatively charged sugar coat / oligosaccharides surrounding external membrane

Front 602

What is the most common membrane protein? How are they characterized?

Back 602

G protein coupled receptors (GPCRs); 7 transmembrane domains

Front 603

What do peripheral membrane proteins do?

Back 603

Often mediate membrane interaction (usually on cytosolic side)

Front 604

What are two examples of peripheral membrane proteins?

Back 604

Spectrin and ankyrin - associate with cytosolic surface of cell as part of a membrane skeleton

Front 605

What are some important examples of G-protein coupled membrane receptors (GPCRs)?

Back 605

- β-adrenergic receptor
- Visual pigment proteins (rhodopsin)
- Na+/K+ ATPase (sodium pump)

Front 606

What are the different types of carrier proteins?

Back 606

- Primary active transporters
- Passive transporters
- Secondary active transporters

Front 607

What are the steps of the Na+/K+ ATPase?

Back 607

1. Transporter binds 3 Na+ inside
2. Phosphorylation of enzyme (from ATP)
3. Transporter releases 3 Na+ to outside and binds 2 K+
4. Dephosphorylation of enzyme
5. Release 2 K+ inside cell
6. Repeat

Front 608

What values do K+ and Na+ need to be extracellularly?

Back 608

[K+] = 4 mM
[Na+] = 145 mM

Front 609

What kind of transporter is the Na+/K+ ATPase?

Back 609

Primary transporter - uses ATP for energy needs

Front 610

What ions does the anion exchange protein exchange?

Back 610

Chloride and bicarbonate

Front 611

Where is the chloride-bicarbonate exchanger found?

Back 611

Red blood cells (RBC)

Front 612

What is the function of the chloride-bicarbonate exchanger?

Back 612

- Increase rate of movement of bicarbonate ions across RBC plasma membrane more than a million fold
- Necessary for gas exchange

Front 613

What kind of transporter is the chloride-bicarbonate exchanger?

Back 613

Antiport transporter

Front 614

When RBCs or other cells are placed in medium of low ionic strength what happens?

Back 614

Hypo-osmotic - take up water and will burst

Front 615

How do you make "membrane ghosts"? What is the purpose?

Back 615

- Put RBCs in low ionic strength medium; cell swells and bursts (leaking all of its insides)
- Put in normal ionic strength medium and plasma membrane reseals
- Now can study properties of RBC plasma membrane

Front 616

How do water molecules transfer through aquaporins?

Back 616

One molecule at a time

Front 617

What technique can be used to determine the diffusion coefficient (D) for lateral movement of lipids in membrane?

Back 617

Fluorescence Recovery After Photobleaching (FRAP):
- Put fluorescent probes on membrane lipids
- Bleach one area with laser
- Watch how long it takes for area to contain fluorescent probes again (due to diffusion)

Front 618

Why is there incomplete recovery during FRAP experiments?

Back 618

Some lipids are immobile

Front 619

What did SDS-PAGE analysis of the RBC Ghosts show?

Back 619

- Glycophorin and anion exchange protein are principle integral membrane proteins
- Peripheral membrane proteins (spectrin, actin) are organized as a membrane skeleton
- Linkage of membrane proteins like anion exchanger to membrane cytoskeleton via linker proteins such as ankyrin
- Band 4.1 greatly restrains lateral mobility of membrane protein

Front 620

What is hereditary spherocytosis?

Back 620

- Gene mutations that result in abnormalities of spectrin, ankyrin, or band 4.1 in membrane skeleton
- Normally biconcave RBC lose their shape and become fragile
- RBC break up as they pass through narrow capillaries
- Leads to hemolytic anemia

Front 621

In hereditary spherocytosis why do the RBC not maintain their biconcave disk shape?

Back 621

- Actin/spectrin network maintains shape
- This disease causes abnormalities in these proteins

Front 622

What is the function of co- and post-translational modifications (PTMs)?

Back 622

Increase functional diversity of a limited gene pool (only 20 AA's)

Front 623

How common is glycosylation?

Back 623

~50% of all proteins undergo some type of glycosylation

Front 624

Why is adding sugars via glycosylation have enormous potential for increasing diversity of proteins?

Back 624

There are not only many different kinds of sugars, but there are many different linkages that can combine them

Front 625

What carbohydrate is generally found in glycolipids?

Back 625

Mannose

Front 626

What are some negatively charged carbohydrates commonly found in mammalian glycoproteins and glycolipids?

Back 626

Sialic acid, glucuronic acid, and iduronic acid

Front 627

What carbohydrates are found predominantly in glycosaminoglycans?

Back 627

Xylose, glucuronic acid, and iduronic acid

Front 628

How must monosaccharides be modified before they can function as immediate precursors in glycoprotein and glycolipid biosynthesis?

Back 628

Monosaccharides must be "activated" to high-energy compounds (i.e., nucleotide sugars)

Front 629

What is the major in vivo source for synthesis of nucleotide sugars?

Back 629

Glucose (Glc)

Front 630

What is necessary to make the early intermediate in the pathway of creating nucleotide sugars? (e.g. sugar 1-phosphate)

Back 630

ATP

Front 631

What is the substrate and donor of sugar residues for the reaction catalyzed by glycogen synthase?

Back 631

UDP-Glucose (UDP-Glc)

Front 632

What is the substrate for several families of glucuronyltransferase?

Back 632

UDP-Glucuronic Acid (UDP-GlcA)

Front 633

What is UDP-Glucuronic Acid (UDP-GlcA) involved in (re: glycosylation)?

Back 633

- Glycoprotein / Proteoglycan synthesis
- Drug detoxification of xenobiotics
- Excretion of steroid hormones
- Heme metabolism and excretion of bilirubin (RBC turnover)

Front 634

What is the function of glycosyltransferases?

Back 634

- Transfer carbohydrate residues from an activated donor substrate, usually a nucleotide sugar, to an acceptor that can be a monosaccharide, an oligosaccharide (glycan), a glycoprotein, or a glycolipid
- Some even transfer sugar to hydroxyl group of some amino acids

Front 635

Where are glycosyltransferases found?

Back 635

Endoplasmic reticulum and Golgi apparatus

Front 636

How do proteins move through the secretory pathway?

Back 636

Transport vesicles through ER, Golgi, and plasma membrane

Front 637

What is the required sequence for an "N-glycosidic" linkage?

Back 637

Asn-X-Ser(Thr)
Sugar binds to N of Asn

Front 638

What residue is bound in an "O-glycosidic" linkage?

Back 638

Serine (sugar binds to hydroxyl group)

Front 639

Preassembly of a glycan (oligosaccharide) chain occurs on which lipid? What kind of lipid is this?

Back 639

Dolichol Phosphate (dolichol-P)
- Membrane-associated
- Isoprenoid derivative
- Precursors: mevalonic acid and cholesterol

Front 640

How is dolichol-P regulated?

Back 640

Regulation of cholesterol metabolism (because cholesterol is a precursor)

Front 641

Which enzyme catalyzes the co-translational transfer of the 14 residue oligosaccharide chain from dolichol-P to Asn residue for an N-linked sugar? Where does this occur?

Back 641

Oligosaccharyltransferase (OST) - takes place in lumen of RER

Front 642

Do all N-linked sugar modifications have the same sugar sequence?

Back 642

No, although they do start with the same 14 residue oligosaccharide structure (Glc3Man9GlcNAc2) this is further processed

Front 643

The final oligosaccharide structure of an N-linked sugar is based on what?

Back 643

The order in which that glycoprotein encountered specific processing glycosyltransferases and glycosidases

Front 644

What is the function of glycosidases?

Back 644

Hydrolytic enzymes that remove carbohydrates

Front 645

The original N-linked oligosacchardie structure is recognized by what? What is their purpose?

Back 645

- Chaperones in the ER called calnexin and calreticulin (lectins)
- Proteins that bind to a specific glycan structure that assist newly synthesized proteins fold into native conformation

Front 646

What are proteoglycans and how do they differ from glycoproteins?

Back 646

- Special class of glycoproteins
- Have specific glycans called glycosaminoglycans

Front 647

What is the structure of proteoglycans?

Back 647

- Protein backbone to which glycosaminoglycan chain(s) are covalently attached
- Glycosaminoglycans are negatively charged polysaccharides composed of repeating disaccharide units
- Disaccharide units consist of an amino sugar (glucosamine or galactosamine) and a uronic acid (glucuronic acid or iduronic acid)
- These sugars may be sulfated

Front 648

How are the glycosaminoglycans attached to proteoglycans?

Back 648

Xyl-Ser O-lnkage

Front 649

What is the purpose of hyaluronic acid (also called "hyaluronan")?

Back 649

Serves as a scaffolding protein for large numbers of proteoglycans in ECM (it is not covalently attached to protein and not sulfated)

Front 650

In what tissues are proteoglycans highly prominent?

Back 650

Connective tissues

Front 651

What are some examples of genetic diseases that are caused by defects in glycan synthesis?

Back 651

- N-glycans (congenital disorders of glycosylation or CDG)
- Glycosaminoglycans (chondrodysplasias)
- O-glycans on α-dystroglycan (at least 5 different muscular dystrophies)

Front 652

What is erythropoietin (EPO)?

Back 652

Growth factor secreted by kidney that stimulates production of RBCs

Front 653

What is erythropoietin (EPO) used to treat?

Back 653

Anemia caused by bone marrow suppression (e.g. after cancer chemotherapy)

Front 654

Describe the structure of erythropoietin (EPO).

Back 654

- 165 residue protein
- Contains N-linked oligosaccharides
- Heavily glycosylated w/ carbohydrates constituting ~40% of mass

Front 655

If erythropoietin (EPO) is not glycosylated, how does it function?

Back 655

Only exhibits 10% of normal activity

Front 656

What is the most highly used drug today? What is its function?

Back 656

Heparin (Lovenox) - anticoagulant

Front 657

What is the functional mechanism of Heparin/Lovenox?

Back 657

- Heparin forms a high affinity complex with antithrombin via Heparin's highly sulfated domains ("NS" domains)
- Antithrombin undergoes a conformational change which increases its activity 1000-10,000 fold
- Antithrombin inhibits two principle procoagulant proteases (factor Xa and thrombin) thereby decreasing the number of fibrin clots

Front 658

How does the function of Heparin/Lovenox compare to Warfarin/Coumadin?

Back 658

Heparin has an immediate impact on coagulation

Front 659

How do proteoglycans function in structural support?

Back 659

- Glycosaminoglycans bind water molecules
- This allows cartilage to resist compression in joints
- "Bubble-packing material"

Front 660

How can the structural support of proteoglycans be utilized to treat osteoarthritis (OA)?

Back 660

- HYALGAN - hyaluronic acid preparation
- Injected into joints
- Helps joint resist compression

Front 661

How can glycoprotein structures be used as "information"?

Back 661

- Cellular recognition
- Intracellular targeting
- Binding sites for bacterial toxins/parasites

Front 662

What are the antigens found on blood cells made of?

Back 662

Antigens are oligosaccharides whose structures are genetically polymorphic (multiple forms)

Front 663

What are changes in cell surface carbohydrates associated with?

Back 663

Cancer cells

Front 664

How is abnormal glycosylation a marker of cancer cells?

Back 664

- Changes in activity or expression of glycosyltransferases and/or glycosidases
- Frequently observed in cancer cells

Front 665

What is Glycation?

Back 665

- Non-enzymatic process that generates glycosylated proteins
- Long-lived proteins of lens, plasma, and RBCs are most susceptible

Front 666

What happens if hemoglobin is "glycated" (HbA1c)?

Back 666

- Glucose becomes covalently attached to ε-amino groups of lysine residues in Hb
- The aldehyde form of glucose first forms a Schiff base w/ NH2 amino groups of HB
- Undergoes an Amadori rearrangement to form a more stable amino ketone linkage
- Ketoamine can further cyclize to yield glycated HbA1c

Front 667

The production of HbA1c (glycated hemoglobin) is dependent upon what?

Back 667

Amount of glucose in blood and duration of hyperglycemia

Front 668

In prolonged hyperglycemia, how is Hb affected?

Back 668

Hb becomes glycated (HbA1c) - this may rise to as high as 12% or more

Front 669

How can the amount of HbA1c be used to monitor diabetic patients?

Back 669

- The amount of HbA1c corresponds to amount of glucose in blood and duration of hyperglycemia
- Used to monitor effectiveness of insulin treatment

Front 670

What are Advanced Glycation End Products (AGEs)?

Back 670

- Heterogenous compounds formed from glycated proteins that are oxidized
- They can damage other proteins by cross-linking them, causing pathological changes

Front 671

How does the influenza virus bind to the host cell?

Back 671

- Influenza A virus is coated w/ several antigenic proteins including hemagglutinin (HA) and sialidase
- HA on virus binds to receptors on host cell, leading to internalization and subsequent membrane fusion events

Front 672

How can avian, swine, and human influenza be distinguished? Why is there a species barrier between avian and human influenza viruses

Back 672

- Avian flus contains α2,3 linked sialic acid on galactose (found predominantly in intestines and respiratory tract of birds)
- Human flus contain α2,6 linked sialic acid on galactose (found predominantly on epithelial cells of human upper respiratory tract)
- Swine flu has both α2,3 and α2,6 linkages

Front 673

What drugs have been developed to treat the flu after onset?

Back 673

- Oseltamivir (Tamiflu) and Zanamivir (Relenza)
- Analogs of sialic acid
- Do not kill virus, but slow replication to a level where immune system can more easily destroy it
- The active site in sialidase is invariant in majority of virus strains (making these effective inhibitors)

Front 674

Where does catabolism of glycolipids, glycoproteins, and proteoglycans occur?

Back 674

Lysosome

Front 675

How does degradation of glycolipids, glycoproteins, and proteoglycans occur?

Back 675

Carbohydrate units are degraded by the sequential action of hydrolytic enzymes (glycosidases) solely from the non-reducing end of the oligosaccharide

Front 676

What are lysosomal storage disorders caused by?

Back 676

- Result from deficiency of a catabolic enzyme and the storage material represents the substrate of the missing enzyme
- Usually a deficiency of a glycosidase

Front 677

If you are unable to cleave a particular carbohydrate linkage, what happens?

Back 677

- Accumulation of that particular substrate in the lysosome
- Cell becomes distorted, inactivated, or destroyed by accumulation

Front 678

When is the protein modification "hydroxylation" particularly important?

Back 678

- Collagen synthesis
- Proline is converted to hydroxyproline (necessary for helix structure)

Front 679

Which enzyme hydroxylates proline for collagen synthesis?

Back 679

Prolyl 4-hydroxylase

Front 680

What protein modification is specific to lysine residues?

Back 680

Acetylation of lysine ε-amino groups (N-acetylation) in histones

Front 681

How does acetylation of lysine residues on histones affect DNA?

Back 681

- Acetylation removes the positive charge on the lysine side chain
- This inhibits the ability of histones, which are rich in lysine and arginine residues to interact w/ negatively charged DNA
- This affects transcription

Front 682

What residues are prone to the protein modification "phosphorylation"?

Back 682

- Serine
- Threonine
- Tyrosine
(all have OH groups)

Front 683

Which types of enzymes phosphorylate proteins?

Back 683

Protein kinases, which use ATP as substrate

Front 684

Which types of enzymes dephosphorylate proteins?

Back 684

Protein phosphatases

Front 685

What is the effect of a phosphorylation modification?

Back 685

Addition of a bulky, negatively charged group into a region of protein that was only moderately polar previously

Front 686

What can be the effects of phosphorylation?

Back 686

- Dramatic change in protein conformation to convert an inactive protein into an active protein
- Serve as a docking/binding site for other proteins in signaling cascades
- Serve as a modulator of the activity of enzymes

Front 687

What is an example of phosphorylation changing the protein conformation?

Back 687

- Insulin receptor
- On / Off switch

Front 688

What is an example of phosphorylation serving as a docking/binding site for other proteins in signaling cascades?

Back 688

- Erythropoietin signaling pathway that stimulates the formation of RBCs

Front 689

What is an example of phosphorylation modulating activity of enzymes?

Back 689

Glycogen synthase has at least 9 different phosphorylation sites, and the pattern of phosphorylation modulates the activity of the enzyme

Front 690

Which protein modification adds a bulky base containing negatively charged phosphates (more than one, not phosphorylation)?

Back 690

ADP-ribosylation
catalyzed by enzymes using NAD+ as substrate

Front 691

Bacterial toxins such as diphtheria, cholera, and pertussis toxins act by modifying crucial host cell proteins with what type of protein modification? Consequences? Example

Back 691

- Mono-ADP-ribosylation
- Critical to pathogenic mechanism that cause these diseases
- Ex: cholera toxin modifies Gs protein rendering it permanently active; ultimately leading to massive water loss, dehydration, and electrolyte loss

Front 692

How are cellular responses to DNA strand breaks in mammalian cells coordinated?

Back 692

By polymerization of ADP-ribose moieties onto substrate proteins carried out by poly ADP-ribose polymerase (PARP)

Front 693

Why is PARP (poly ADP-ribose polymerase) a cancer drug target?

Back 693

- Involved in DNA damage repair and maintaining genome stability
- Responses to DNA strand breaks is coordinated by polymerization of ADP-ribose moieties

Front 694

What are the purposes of protein degradation?

Back 694

1. Proteins that are improperly folded, mis-assembled, or damaged will be removed, preventing accumulation
2. Mechanism for maintaining the appropriate levels of proteins and for permitting rapid changes in levels so that cell can adapt to conditions

Front 695

What are the two major protein degradation pathways in mammalian cells?

Back 695

1) proteasome
2) lysosome

Front 696

What molecule tags proteins for degradation?

Back 696

Ubiquitin

Front 697

Which accessory protein helps ubiquitin identify which proteins should be tagged for degradation?

Back 697

E3

Front 698

What are the three helper proteins to ubiquitin in targeting protein degradation?

Back 698

- E1 = activates ubiquitin to form thioester in ATP dependent process
- E2 = ubiquitin is transferred to E2 enzymes (ubiquitin conjugating enzyme)
- E3 = links ubiquitin to target proteins (ubiquitin ligase)

Front 699

How is Ubiquitin linked to target proteins?

Back 699

- Isopeptide bond between:
- ε-amino group of lysine residues of target protein and
- carboxyl-terminal glycine residue of ubiquitin

Front 700

When proteins are ubiquitinated, what is their fate?

Back 700

Degraded by a large complex called a proteosome

Front 701

What does it mean to be ubiquitinated (must be polyubiquitinated, not monoubiquinated)?

Back 701

- Contain a linear, polyubiquitin chain
- Lined up for degradatio by proteasome in cytosol

Front 702

How does the proteasome degrade proteins?

Back 702

- Hydrolyzes ATP to unfold proteins prior to proteolysis by inner chamber of proteasome

Front 703

What are the consequences of a protein being only "monoubiquitinated"?

Back 703

- Not targeted to proteasome for degradation
- Some proteins are triggered to undergo endocytosis and are subsequently delivered to lysosome for degradation

Front 704

What is an example of a defect in the ubiquitin-proteasome system for protein degradation?

Back 704

- Heterozygous mutations in BRCA1 (Breast cancer susceptibility locus) are seen in some cases (~10%) of breast and ovarian cancer

Front 705

What is BRCA1?

Back 705

- Breast cancer susceptibility locus
- Heterozygous mutations are seen in 10% of cases of breast and ovarian cancer
- BRCA1 protein is an E3 ubiquitin ligase whose activity is abolished by mutations found in familial breast and ovarian cancers

Front 706

What can inhibit degradation of proteins?

Back 706

- Acetylation of lysine residues on E3 protein
- E3 protein usually attaches a ubiquitin molecule to lysine

Front 707

What is the common function of hydrophobic protein modifications?

Back 707

Promotes interaction of proteins w/in lipid bilayer

Front 708

What are two fatty acid examples of hydrophobic protein modifications?

Back 708

1. Palmitoylation - palmitic acid (C16) is linked through a thioester bond to cysteine
2. Myristoylation - myristic acid (C14) is linked to amino-terminal glycine

Front 709

What happens during farnesylation (C15)?

Back 709

- Farnesyltransferase uses farnesyl pyrophosphate as substrate
- Enzyme requires CAAX consensus sequence at C-terminus of target protein (C=cys, A=aliphatic, X=any aa)
- Farnesylation of cysteine occurs following proteolytic removal of AAX resulting in cys at C-terminus

Front 710

What is an important example of the farnesylation (C15) protein modification?

Back 710

Farnesylation of Ras is required for its association w/ plasma membrane, its functioning in signal transduction, and essential for transforming activity of oncogenic variants

Front 711

Which clinical correlation relates to farnesylation?

Back 711

- Hutchinson-Gilford Progeria Syndrome
- Aging-like phenotypes as children (avg death at 13)
- Caused by accumulation of mutant form of prelamin A (retains a farnesyl group rather than being converted to mature lamin A which lacks farnesyl group)

Front 712

What is the structure of a GPI linkage?

Back 712

- GPI = glycosyl phosphatidylinositol
- GPI is attached to C-terminus of target protein through a phosphoethanolamine portion of molecule
- Two fatty acid moieties of GPI are imbedded in bilayer

Front 713

How does the cell establish and maintain compartmentalization?

Back 713

Selective transport molecules to various organelles; often using a "tag" that is read by specific receptors

Front 714

What is the name of the space between the membrane-bound organelles and takes up ~50% of cell volume?

Back 714

Cytosol

Front 715

What is inside of the cytosol?

Back 715

- Enzymes involved in intermediary metabolism
- Ribosomes

Front 716

What is the purpose of the ribosomes found on the endoplasmic reticulum (ER)?

Back 716

- Actively synthesizing
- Remove selected proteins from cytosol
- Either transmembrane proteins or water soluble proteins

Front 717

How does the cell establish and maintain compartmentalization?

Back 717

Selective transport molecules to various organelles; often using a "tag" that is read by specific receptors

Front 718

How do transmembrane proteins and water soluble proteins differ?

Back 718

- Transmembrane - only partly translocated across ER membrane and become embedded
- Water soluble - fully translocated across ER and released into lumen

Front 719

What is the name of the space between the membrane-bound organelles and takes up ~50% of cell volume?

Back 719

Cytosol

Front 720

What are the two types of water soluble proteins?

Back 720

- Secretory proteins
- Proteins destined for lysosome

Front 721

What is inside of the cytosol?

Back 721

- Enzymes involved in intermediary metabolism
- Ribosomes

Front 722

Where does all protein synthesis start?

Back 722

Cytosolic ribosomes

Front 723

What is the purpose of the ribosomes found on the endoplasmic reticulum (ER)?

Back 723

- Actively synthesizing
- Remove selected proteins from cytosol
- Either transmembrane proteins or water soluble proteins

Front 724

Where does protein synthesis finish?

Back 724

- Cytosolic ribosomes
- Ribosomes attached to ER (for transport into membrane or into lumen)

Front 725

How do transmembrane proteins and water soluble proteins differ?

Back 725

- Transmembrane - only partly translocated across ER membrane and become embedded
- Water soluble - fully translocated across ER and released into lumen

Front 726

Ribosomes that finish synthesis in the cytosol can go where ultimately?

Back 726

- Mitochondria
- Nucleus
- Peroxisomes

Front 727

What are the two types of water soluble proteins?

Back 727

- Secretory proteins
- Proteins destined for lysosome

Front 728

Ribosomes that finish synthesis on/in the ER can go where ultimately?

Back 728

- First to Golgi
- Lysosomes or secretory vesicles or cell surface

Front 729

Where does all protein synthesis start?

Back 729

Cytosolic ribosomes

Front 730

Where does protein synthesis finish?

Back 730

- Cytosolic ribosomes
- Ribosomes attached to ER (for transport into membrane or into lumen)

Front 731

Ribosomes that finish synthesis in the cytosol can go where ultimately?

Back 731

- Mitochondria
- Nucleus
- Peroxisomes

Front 732

Ribosomes that finish synthesis on/in the ER can go where ultimately?

Back 732

- First to Golgi
- Lysosomes or secretory vesicles or cell surface

Front 733

How are proteins targeted to the ER membrane?

Back 733

- Signal sequence on protein
- Recognized by Signal Recognition Particle (SRP)
- SRP binds signal sequence and ribosome
- SRP binds to SRP receptor on ER membrane and ribosome binds to adjacent receptors
- Peptide translocation complex sends polypeptide into ER lumen
- Signal peptidase cleaves signal sequence
- Protein folds inside lumen (SRP binding prevented protein from folding until in ER)

Front 734

What is the structure of the signal recognition particle (SRP) that recognizes the signal sequence targeting a protein to the ER?

Back 734

- Ribonucleoprotein
- Single 7SL RNA molecule plus 6 different polypeptide chains

Front 735

What are the functions of the signal recognition particle (SRP)?

Back 735

- Recognizes signal sequence on growing polypeptide
- Arrests elongation so that it may translocate across membrane

Front 736

Where is the signal recognition particle (SRP) located?

Back 736

Cycles between ER membrane and cytosol

Front 737

What is the structure of the signal sequence that targets a protein for the ER?

Back 737

- No consensus sequence
- Usually 13-48 residues
- Usually occurs at amino terminus
- Tripartite domain structure: hydrophilic amino-terminal domain w/ net positive charge, hydrophobic core domain w/ α-helix, and a polar carboxyl-terminal domain
- Different primary sequence but similar 3D conformation

Front 738

What removes the signal sequence from the protein once it has successfully gotten to/in the ER?

Back 738

Signal peptidase

Front 739

Where is the SRP receptor located?

Back 739

- Exclusively in ER
- Integral membrane protein w/ SRP binding site exposed to cytosol

Front 740

What is the function of the SRP receptor?

Back 740

Binds the SRP on the nascent polypeptide/ribosome complex, effectively targeting complex to ER membrane

Front 741

What is the function of the ribosome receptor?

Back 741

Binds to the ribosome and stabilizes the ribosome/nascent polypeptide chain complex on the ER membrane

Front 742

What is the function of signal peptidase

Back 742

- Integral membrane protease w/ active site facing lumen of ER
- Cleaves amino-terminal signal sequences co-translationally

Front 743

What happens during the translocation process across ER membrane?

Back 743

- ATP hydrolysis required
- Occurs through aqueous pore or channel called "translocon"
- Translocation and protein synthesis are usually coupled since unfolded polypeptide chains are preferred
- Translocated polypeptide chains fold in lumen of ER w/ assistance of chaperones

Front 744

What are "stop-transfer sequences"?

Back 744

Hydrophobic, α-helical sequences which function to anchor the protein in membrane

Front 745

If a protein traverses the membrane multiple times, how many stop-transfer sequences will it have?

Back 745

Multiple sequences to tell protein to cross membrane again

Front 746

How are transmembrane portions of a polypeptide sequence anchored into the membrane?

Back 746

Hydrophobic interactions between non-polar residues and fatty acyl groups of membrane

Front 747

What is the structure of the Golgi?

Back 747

- 4-12 cisternae
- Two distinct sides: cis face is juxtaposed to ER and trans face is distended into a tubular reticulum called the trans Golgi network (TGN)

Front 748

What happens in the trans Golgi network (TGN)?

Back 748

- Site in cell where proteins that have been synthesized on ER-bound ribosomes are sorted for transport
- Go to lysosomes, secretory granules, or plasma membrane

Front 749

What happens in the constitutive pathway?

Back 749

- Common to all cells
- Soluble proteins are continuously secreted without intracellular storage

Front 750

What happens in the regulated pathway?

Back 750

- Only in certain cells (exocrine, endocrine, and neurons)
- Subset of secretory proteins is sorted to storage organelles (called secretory granules) from which they are released only upon stimulation

Front 751

What kind of cells have secretory granules? Why?

Back 751

- Exocrine cells
- Endocrine cells
- Neurons
- Allows contents to be released only upon stimulation

Front 752

Which organelle is like a "recycling center"?

Back 752

Lysosome

Front 753

What is the function of the lysosome?

Back 753

- Disposal of abnormal proteins
- Downregulation of cell surface signaling receptors
- Release of endocytosed nutrients
- Degradation of pathogenic organisms (phagocytosis)
- Cellular survival (autophagy)

Front 754

What kind of enzymes are found in the lysosome?

Back 754

- Acid hydrolases (hydrolytic enzymes)
- Optimally active at pH of 5

Front 755

How is the pH of 5 maintained in the lysosome?

Back 755

V-type ATPases (V for vacuolar) couple ATP hydrolysis to transport of protons into lysosome (and endosomes)

Front 756

What is autophagy?

Back 756

- "Self-eating"
- Major catabolic, energy-producing pathway in eukaryotes
- Involves lysosomal degradation of cytoplasmic proteins and organelles and protein aggregates

Front 757

What is the major catabolic, energy producing pathway in eukaryotes?

Back 757

Autophagy (self-eating)

Front 758

What is the function of autophagy?

Back 758

- Eliminate damaged organelles and protein aggregates
- During starvation it dramatically increases and provides internal source of nutrients for energy generation and survival

Front 759

Intracellular protein aggregates and dysfunctional organelles are common features of what types of diseases?

Back 759

Neurodegenerative diseases:
- Alzheimer's
- Parkinson's
- Huntington's
- Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's)

Front 760

What happens in a lot of neurodegenerative diseases?

Back 760

- Toxic protein aggregates and damaged organelles accumulate within specific types of neurons
- Leads to neuronal dysfunction and ultimately neuronal death

Front 761

What is a potential therapeutic target of neurodegenerative diseases?

Back 761

Regulation of autophagic pathways (to degrade protein aggregates and dysfunctional organelles)

Front 762

The proteins found in lysosomes are synthesized where?

Back 762

- On ribosomes bound to ER membrane so that they can be transported through Golgi apparatus
- Requires that they have a signal sequence

Front 763

How are soluble acid hydrolases targeted to the lysosome?

Back 763

- Tagged with mannose 6-phosphate
- Directed to lysosome by mannose 6-phosphate receptors

Front 764

What are 3 components of lysosomal enzyme targeting?

Back 764

- GlcNAc phosphotrnasferase - recognizes a protein signal on lysosomal enzymes and transfers GlcNAc-1P to specific mannose residues
- GlcNAc glycosidase (phosphodiesterase) - cleaves off GlcNAc residue generating mannose 6-P recognition marker
- Mannose 6-P receptors - recognizes mannose 6-P on lysosomal enzymes and transports them from Golgi to prelysosomal compartment (endosomes)

Front 765

What is the name for the general disease of lysosome function? What is it usually caused by?

Back 765

- Lysosomal Storage Disorders (LSDs)
- Defective activity of a single lysosomal hydrolytic enzyme

Front 766

What are the effects of a Lysosomal Storage Disorder (LSD)?

Back 766

- Intra-lysosomal accumulation of undegraded substrates
- Leads to enlarged lysosomes ("inclusion bodies")
- Leads to progressive multi-organ involvement (neurological, liver, spleen, heart, kidney, abnormal bone formation, muscle atrophy, ocular disease)

Front 767

What kind of therapies are there for Lysosomal Storage Disorders (LSDs)?

Back 767

- Enzyme Replacement Therapy (ERT)
- Dependent upon receptor-mediated endocytosis of mannose 6-P tagged recombinant acid hydrolases via cell surface mannose 6-P receptors

Front 768

What are some examples of Lysosomal Storage Disorders (LSDs)?

Back 768

- Gaucher's
- Fabry's
- Hurler's
- Pompe's disease

Front 769

What causes mucolipidosis II (I-cell disease)?

Back 769

Defects in lysosomal enzyme targeting because of a deficiency in the enzyme (GlcNAc phosphotransferase) that generates mannose 6-phosphate tag on proteins destined for lysosome

Front 770

What are the consequences of mucolipidosis II (I-cell disease)?

Back 770

- Cells lack multiple lysosomal enzymes in lysosomes and abnormally high levels of lysosomal enzymes in plasma and other body fluids
- Severe psychomotor retardation, hepatosplenomegaly (enlargement of spleen and liver), many skeletal abnormalities, coarse facial features, and restricted joint movement
- Usually die by age 10

Front 771

What does vesicular transport between organelles involved?

Back 771

- Coat protein-covered vesicle - buds from donor compartment
- Coat proteins released from vesicle
- Uncoated vesicle binds to a specific target protein
- Fusion of uncoated vesicle to target membrane

Front 772

How are SNARES involved in vesicular fusion?

Back 772

- Integral membrane proteins
- Each vesicle has its own v-SNARE which matches to a t-SNARE found on target membrane (ensures specificity)

Front 773

What are the steps of vesicular fusion with SNARE molecules?

Back 773

- Vesicle approaches plasma membrane of target
- v-SNARE and t-SNARE bind to each other, zipping up from amino termini and drawing two membranes together
- Zipping causes curvature and lateral tension on bilayers, favoring hemifusion between outer leaflets
- Hemifusion: inner leaflets of both membranes come in contact
- Complete fusion creates a fusion pore
- Pore widens; vesicle contents are released outside cell

Front 774

What are the effects of the Tetanus toxin released by Clostridium?

Back 774

- Blocks release of inhibitory neurotransmitter γ-aminobutyric acid (GABA)
- Results in a spastic paralysis ("lockjaw")

Front 775

What are the effects of the Botulinum toxin released by Clostridium?

Back 775

- Blocks release of Acetylcholine (ACh)
- Results in flaccid paralysis

Front 776

What was "Botox" (diluted botulinal toxin) originally approved for?

Back 776

- Crossed eyes
- Eyelid tics

Front 777

What is Botox (diluted botulinum toxin) used for commonly?

Back 777

- Overactive muscle contraction
- Brow wrinkling
- Back pain
- Bladder spasms
- Migraine headaches
- Writer's cramp
- Incontinence
- Patients w/ cerebral palsy

Front 778

How do Tetanus and Botulinum toxins function molecularly?

Back 778

- Peptidases that cleave SNAREs essential for synaptic vesicle fusion to plasma membrane
- Tetanus targets GABA release
- Botulinum targets ACh release

Front 779

What is "endocytosis"?

Back 779

Internalization of material from the external environment enclosed within a membrane derived from the plasma membrane

Front 780

What are the steps of Receptor-mediated endocytosis?

Back 780

- Macromolecules bind to complementary cell-surface receptors and accumulate in coated pits
- Enter the cell as receptor-macromolecule complexes in endocytic vesicles
- Endocytic vesicles fuse with endosomes where receptors and their ligands may be sorted

Front 781

How do the endosomes become acidic?

Back 781

V-type ATPases (although not as acidic as lysosomes)

Front 782

What occurs in endosomes?

Back 782

- Receptors and ligands are sorted from one another
- Selective concentrating mechanism to increase efficiency of internalization of particular ligands

Front 783

What are the four pathways of receptor-mediated endocytosis (what can happen after the receptor and macromolecule are inside the endosome)?

Back 783

- Receptor recycled and ligand degraded
- Receptor and ligand recycled
- Receptor and ligand degraded
- Receptor and ligand transported

Front 784

What is the major protein component of coated pits/vesicles?

Back 784

Clathrin - triskelion-shaped "3-legged" protein complex is composed of noncovalently associated polypeptide chains (3 heavy and 3 light chains); chains undergo self-assembly to form polyhedral protein baskets/cages

Front 785

How much of the plasma membrane surface is covered in coated pits of clathrin?

Back 785

2%

Front 786

What is required before the vesicle can fuse to the endosome?

Back 786

Removal of clathrin coat

Front 787

How can iron be transported into the cells?

Back 787

Via transferrin / transferrin cycle

Front 788

What is the transferrin cycle?

Back 788

- Iron binds to transferrin
- Transferrin bound to iron binds to transferrin receptor
- Clathrin-coated pit buds off to form clathrin-coated vesicle
- Clathrin removed and turns into endosome
- Proton pump (V-type ATPase) acidifies endosome
- Iron is released from endosome to make heme
- Endosome fuses with membrane to obtain more iron (via SNARES)

Front 789

What does familial hypercholesterolemia result from?

Back 789

Mutation that affects the structure and function of the LDL receptor

Front 790

What is familial hypercholesterolemia (FH) characterized by?

Back 790

- Elevated conc. of low density lipoprotein (LDL) in plasma
- Deposition of cholesterol in tendons and skin (xanthomas) and in arteries (atheromas)
- Inheritance as an autosomal dominant trait w/ a gene dosage effect (homozygotes are more severely affected)

Front 791

What is one of the most common inborn errors of metabolism?

Back 791

Familial hypercholesterolemia (FH) (heterozygotes 1 in 500 persons; homozygotes 1 in 1 million)

Front 792

What are the consequences of familial hypercholesterolemia (FH)?

Back 792

Coronary heart disease begins in childhood

Front 793

How can the mutations of the LDL receptor that causes familial hypercholesterolemia (FH) be classified?

Back 793

5 classes:
- 1 = defect in synthesis
- 2 = defect in transport
- 3 = defect in binding
- 4 = defect in clustering
- 5 = defect in recycling

Front 794

Signaling receptors are what kind of proteins? What is their function?

Back 794

- Transmembrane proteins on cell surface; upon binding of ligand they become activated and generate various intracellular signals
- Also intracellular receptors which are stimulated by small hydrophobic signal molecules that can diffuse across membrane

Front 795

What are the four types of signaling molecules?

Back 795

- Contact-dependent
- Paracrine
- Synaptic
- Endocrine

Front 796

When is contact dependent signaling particularly important/utilized?

Back 796

Development and in immune responses

Front 797

When does paracrine signaling occur?

Back 797

- Between different cell types
- Called autocrine signaling if same cell type
- Cancer cells often rely on autocrine signaling for survival and proliferation

Front 798

What is the function of synaptic signaling?

Back 798

Coordinates behavior of cells in remote parts of body; very fast

Front 799

What does endocrine signaling depend upon?

Back 799

Diffusion and blood flow; relatively slow

Front 800

Describe what kind of cell signaling changes are "slow" responses?

Back 800

- Increased cell growth
- Changes in gene expression
- Changes in protein synthesis

Front 801

Describe what kind of cell signaling changes are "fast" responses?

Back 801

- Cell movement
- Cell secretion
- Metabolism (rapid phosphorylation of effector proteins)

Front 802

The same signal can mediate many different effects, how does this happen? Example?

Back 802

- Different receptor proteins
- Different intracellular machinery
- Heart muscle and salivary gland cells use same ACh receptor, but ACh induces different cellular responses (due to difference in intracellular machinery)

Front 803

How is rapid relaxation of smooth muscle cells coordinated?

Back 803

- Autonomic nerves in vessel wall release ACh
- ACh acts on nearby endothelial cells by stimulating NO synthesis
- NO diffuses across membrane and binds to guanylyl cyclase in smooth muscle cells
- Activated guanylyl cyclase converts GTP to cGMP
- cGMP induces rapid relaxation of smooth muscle cells

Front 804

Intracellular receptors are structurally related and belong to what very large superfamily? What structural feature do they have?

Back 804

Nuclear Receptor Superfamily - contain DNA binding domains that regulate transcription

Front 805

How do most signaling ligands that bind to intracellular nuclear receptors enter the cell?

Back 805

Via carrier proteins for transport in blood stream; dissociate from carrier proteins when they enter into cells

Front 806

When steroid hormone receptors translocate into the nucleus upon ligand binding, what happens?

Back 806

Ligand binding induces conformational changes in nuclear receptors and affects their interaction with either inhibitory proteins or coactivator proteins; this determines whether they regulate transcription positively or negatively

Front 807

Direct stimulation of early response gene expression can occur how quickly?

Back 807

In 30 minutes

Front 808

What is the "delayed response"?

Back 808

The gene products from the "early response" induce expression of other genes "delayed response"

Front 809

What are the roles of the early response gene products?

Back 809

- Induce expression of delayed response genes
- Negative feedback on primary response genes

Front 810

Why is nuclear receptor signaling usually specific?

Back 810

- Only certain types of cells have receptors
- Each of these cell types contains a different combination of other cell-type-specific gene regulatory proteins that collaborate w/ activated receptor to influence transcription of specific genes

Front 811

What are the three largest classes of cell surface receptors?

Back 811

- Ion-channel coupled receptors
- G-protein coupled receptors
- Enzyme coupled receptors

Front 812

What are ion-channel coupled receptors involved in?

Back 812

Rapid synaptic signaling (aka transmitter-gated channels or ionotropic receptors)

Front 813

What are G-protein coupled receptors involved in?

Back 813

Indirectly regulate plasma membrane-bound enzymes or ion channels, which is mediated by a trimeric GTP-binding protein

Front 814

What are enzyme coupled receptors involved in?

Back 814

Function as enzymes or associate directly with enzymes that they activate

Front 815

What do "molecular switch" proteins do?

Back 815

Relay signal to next signaling component in chain

Front 816

What do "scaffold proteins" do?

Back 816

Bring two or more signaling proteins together so that they can interact more quickly and efficiently

Front 817

Primary signals are transduced to produce large amounts of what?

Back 817

Large amounts of small intracellular mediators or many copies of a downstream signaling protein, which amplifies the signal

Front 818

What is a signaling cascade?

Back 818

When there are multiple amplification steps in a relay chain

Front 819

What is a coincidence detector?

Back 819

A molecule that receives signals from two or more signaling pathways and integrates them before relaying a signal onward

Front 820

What would be the purpose of a protein anchor in a cell signaling pathway?

Back 820

Some proteins anchor one or more signaling proteins in a pathway to a particular structure in cell where signaling proteins are needed

Front 821

What are examples of "molecular switches"?

Back 821

- Phosphorylation
- GTP-binding
- (also cAMP-binding, Ca2+-binding, and protein modifications such as ubiquitylation)

Front 822

When a G-protein has GTP bound is it on or off? How can it modify this?

Back 822

On - intrinsic GTPase activity to shut themselves off by hydrolyzing their bound GTP to GDP

Front 823

What are the two types of GTP-binding proteins in cells?

Back 823

- Monomeric GTP-binding proteins (small GTPases)
- Trimeric GTP binding proteins (G proteins)

Front 824

What is the function of GTPase-activating proteins (GAPs)?

Back 824

Increase rate of GTP hydrolysis of small GTPases (leads to inactivation)

Front 825

What do many receptor tyrosine kinases do?

Back 825

Generate specific docking sites on themselves to interact with signaling proteins

Front 826

What do phosphoinositides do?

Back 826

Generate docking sites in plasma membrane to recruit intracellular signaling proteins

Front 827

What are some examples of highly conserved small interaction domains? What do they bind to?

Back 827

- Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains bind to phosphorylated tyrosine
- Src homology 3 domains bind short proline rich amino acid sequences
- Pleckstrin homology (PH) domains bind to charged head groups of specific phosphoinositides

Front 828

What interaction domains bind to phosphorylated tyrosine?

Back 828

SH2 (src homology 2) and PTB (phosphotyrosine-binding)

Front 829

What interaction domains bind to short-proline rich amino acid sequences?

Back 829

SH3 (src homology 3)

Front 830

What interaction domains bind to charged head groups of specific phosphoinositides?

Back 830

Pleckstrin homology (PH)

Front 831

What is desensitization / adaptation?

Back 831

Prolonged exposure to a stimulus decreases the cell's response to the level of stimulus

Front 832

What are some different methods of achieving desensitization/adaptation?

Back 832

- Receptor sequestration
- Receptor down-regulation
- Receptor inactivation
- Inactivation of signaling protein
- Production of inhibitory protein

Front 833

Which coupled receptor mediates most cellular responses to signals?

Back 833

G-protein coupled receptors (GPCRs)

Front 834

What kind of signal molecules are there for GPCRs?

Back 834

- Proteins
- Small peptides
- Derivatives of amino acids and fatty acids
- Photon of light
- Smell and taste

Front 835

What is the structure of GPCRs?

Back 835

- Single peptide chain
- 7 transmembrane domains

Front 836

What is a guanine nucleotide exchagne factor (GEF)?

Back 836

Molecule that exchanges GDP for GTP, thus activating the α subunit

Front 837

What is a regulator of G-protein signaling (RGS)?

Back 837

- Increases GTPase activity
- Counterparts of GAPs that regulate small GTPases

Front 838

What are the three G-proteins we need to know and what are their functions?

Back 838

- Gs = (stimulatory) activates adneylyl cyclase, activates Ca2+ channels
- Gi = (inhibitory) inhibits adenylyl cyclase
- Gq = activates phospholipase C-β

Front 839

What is the steps of activating Gs protein?

Back 839

- Signal molecule activates GPCR
- GEF (guanine nucleotide exchange factor) activates α subunit by exchanging GDP to GTP
- Gs-α activates adenylyl cyclase
- AC converts ATP to cAMP
- cAMP binds to 2 regulatory subunits of Protein Kinase A, releasing 2 catalytic PKA subunits
- Activated PKA phosphorylates CREB
- CREB-binding protein (CBP) binds CREB
- Gene transcription is activated

Front 840

How does Cholera toxin affect the Gs-protein?

Back 840

- Cholera toxin transfers ADP-ribose from NAD+ to α-subunit of Gs
- Inhibits GTP hydrolysis
- Sustains active conformation of Gs and subsequently sustains adenylyl cyclase
- Leads to prolonged cAMP production in intestinal epithelial cells leading to large efflux of Cl- and water into gut
- Causes diarrhea

Front 841

How does Pertussis toxin affect the Gs-protein?

Back 841

- Pertussis toxin catalyzes ADP-ribosylation to α subunit of Gi
- Prevents protein from interacting with receptors
- As a result, G protein remains in GDP-bound form
- Unable to regulate target proteins
- Whooping cough

Front 842

What are some responses to increases in cAMP?

Back 842

- Thyroid gland: thyroid hormone synthesis and secretion via TSH
- Adrenal cortex: cortisol secretion via ACTH
- Ovary: progesterone secretion via LH
- Muscle: glycogen breakdown via adrenaline
- Bone: bone resorption via parathormone
- Heart: increase in HR and force of contraction via adrenaline
- Liver: glycogen breakdown via glucagon
- Kidney: water resorption via vasopressin
- Fat: triglyceride breakdown via adrenaline, ACTH, glucagon, and TSH

Front 843

What is the function of phospholipase C-β? How is it activated?

Back 843

- Activated by Gq-protein
- Cleaves phosphotidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)

Front 844

How does phospholipace C-β activation by Gq-protein ultimately lead to activation of Protein Kinase C?

Back 844

- Phospholipase C cleaves PIP2 into IP3 and DAG
- IP3 induces release of Ca2+ from ER
- DAG and Ca2+ activate protein kinase C (PKC)

Front 845

What are some of the major responses to PLC-β?

Back 845

- Liver: glycogen breakdown via vasopressin
- Pancreas: amylase secretion via ACh
- Smooth muscle: muscle contraction via ACh
- Blood platelets: platelet aggregation via thrombin

Front 846

What is another function of DAG besides mediating activation of PKC?

Back 846

Mediates pain and inflammatory responses by regulating synthesis of prostaglandins (which is inhibited by most anti-inflammatory drugs)

Front 847

What modification is performed to desensitize GPCR proteins?

Back 847

GPCRs are phosphorylated by PKA, PKC or GPCR kinases (GRKs) indicating feedback regulation

Front 848

What is the function of Arrestin?

Back 848

- Prevents phosphorylated GPCR from interacting w/ G proteins
- It also couples GPCR to clathrin-dependent endocytosis machinery

Front 849

What five principle classes of enzyme-coupled receptors are used in humans?

Back 849

1. Receptor tyrosine kinases
2. Tyrosine kinase-associated receptors
3. Receptor Ser/Thr kinases
4. Receptor guanylyl cyclases
5. Receptorlike tyrosine phosphatases

Front 850

What is the function of Receptor tyrosine kinases (RTKs)?

Back 850

- Directly phosphorylate specific tyrosines on themselves and on a small set of intracellular signaling proteins
- Ligand binding causes receptor chains to dimerize, bringing the kinase domains of two receptor chains together so they can become activated and cross-phosphorylate each other (transautophosphoryation)
- Tyr phosphorylation increases kinase activity
- Tyr phosphorylation creates high-affinity docking sites for binding of specific signaling proteins w/ SH2 or PTB domains

Front 851

What is the function of Tyrosine kinase-associated receptors?

Back 851

- Directly recruit cytoplasmic kinases to relay signals
- No intrinsic kinase activity
- Ex: Cytokine receptors mediate signaling via (Janus Kinase) JAK-STAT pathway

Front 852

What is the function of Receptor Ser/Thr kinases? Example?

Back 852

- Directly phosphorylate specific Ser/Thr residues on themselves and proteins they associate with
- Transforming growth factor-β (TGFβ) family members mediate development, immune responses, and cell proliferation and death

Front 853

What is the function of receptor guanylyl cyclases?

Back 853

- Directly catalyze production of cGMP

Front 854

What is the function of receptorlike tyrosine phosphatases?

Back 854

Remove phosphate groups from tyrosine of specific intracellular signaling proteins; they are receptor-like because their ligands are not known

Front 855

What is an important example and exception to the normal rules of receptor tyrosine kinases (RTKs)?

Back 855

- Insulin receptor, IGF1 receptor (ligand binding rearranges their transmembrane receptor chains, moving the two kinase domains close together)
- Do not generate docking sites on the receptor but use a specialized docking protein called insulin receptor substrate 1 (IRS1)

Front 856

What are some functions of Ras proteins (small GTPase)?

Back 856

- Molecular switch for RTK signaling
- Relays signals from RTKs