Biochemistry Review 1 (12/6/08)

Front 1

Nuclear Receptor Ligands include:

Back 1

* CORTICOIDS
1. Glucocorticoids (ie cortisol--increase blood sugar, anti-inflammatory action.)
2. Mineralocorticoids (maintain salt/water balance)

* STEROID SEX HORMONES:
1. Vitamin D3
2. Testosterone

* DEVELOPMENT/MORPHOGENESIS
1. Vitamin D3
2. Triiodothyronine (T3)
3. (trans)retinoic acid

Front 2

Mechanism of action of the nuclear receptor ligands:

Back 2

1. The plasma bound steroid hormone must free itself to become "free steroid hormone" where it enters either the cytosol OR nucleus to bind to either the Hsp-bound non-DNA binding cytoplasmic reeptor or to the nuclear receptor respectively.

3. In the cytosol, binding of the steroid hormone causes dissociation of the Hsp-protein and subsequent translocation of cytoplasmic receptor complex into the nucleus. The cytoplasmic receptor complex is activated following Hsp-protein dissociation.

4. In the nucleus: the free steroid hormone binds to the nuclear receptor leading to activation.

5. The activated nuclear receptor (either from the cytosol or nucleus at this point--the common step) then acts as a transcription factor and promotes mRNA transcription, subsequent translation of mRNA into protein, and the new protein then can leave the cell to promote a biological response.

Front 3

Domains of the steroid receptors.

Back 3

1. Variable domain: Begins @ N-terminal, is the most variable domain between the different receptors.

2. DNA binding domain: This centrally located highly conserved DNA binding domain (DBD) consists of two non-repetitive globular motifs (PDB: 1HCQ) where zinc is coordinated with four cysteine and no histidine residues. Their secondary and tertiary structure is distinct from that of classic zinc fingers.[2] This region controls which gene will be activated. On DNA it interacts with the hormone response element (HRE).

3. Nuclear localization signal (Hinge region): This area controls the movement of the receptor to the nucleus.

3. Transcription activation subdomain: The moderately conserved ligand-binding domain (LBD) can include a nuclear localization signal, amino-acid sequences capable of binding chaperones and parts of dimerization interfaces. Such receptors are closely related to chaperones (namely heat shock proteins hsp90 and hsp56), which are required to maintain their inactive (but receptive) cytoplasmic conformation. At the end of this domain is the C-terminal. The terminal connects the molecule to its pair in the homodimer or heterodimer. It may affect the magnitude of the response.

So in summary:
(N-terminus)
1. Variable (immunogenic)
2. DNA (zinc fingers)
3. Nuclear localization signal
4. Transcription activation subdomain
5. HSP binding site
6. Steroid
(C-terminus)

Front 4

Where on the DNA do steroid receptors bind? What is the effect of this?

Back 4

Steroid receptors + ligand binds to STEROID RESPONSE ELEMENTS located in the regulatory region of the gene, where they can alter the rate of gene transcription.

The steroid receptor binds to the "steorid response element." Then TATA binding protein binds to the TATA box, DNA unwinds, (rich A and T sequences), and then RNA pol II begins transcription of DNA--> mRNA-->protein.

Front 5

TATA box

Back 5

The TATA box has the core DNA sequence 5'-TATAAA-3' or a variant, which is usually followed by three or more adenine bases. It is usually located 25 base pairs upstream to the transcription site. The sequence is believed to have remained consistent throughout much of the evolutionary process, possibly originating in an ancient eukaryotic organism.

It is normally bound by the TATA Binding Protein (TBP) in the process of transcription, which unwinds the DNA, and bends it through 80°. The AT-rich sequence facilitates easy unwinding (due to 2 hydrogen bonds between bases as opposed to 3 between GC pairs). The TBP is an unusual protein in that it binds to the minor groove and binds with a β sheet.

The TATA box is usually found as the binding site of RNA polymerase II. The transcription factor TFIID binds to the TATA box, followed by TFIIA binding to the upstream part of TFIID. TFIIB can then bind to the downstream part of TFIID. The polymerase can then recognise this multi-protein complex and bind to it, along with various other transcription factors such as TFIIF, TFIIE and TFIIH. Transcription is then initiated, and the polymerase moves along the DNA strand, leaving TFIID and TFIIA bound to the TATA box. These can then facilitate the binding of additional RNA polymerase II molecules.

Front 6

In patients with thyroid hormone resistance, what is the molecular nature of their resistance?

Back 6

There is a steroid-binding defect (localized in the steroid domain of the steroid hormone receptor). (francisco was the patient dr. smith used to discuss this illness)

Front 7

G protein coupled receptors

Back 7

G protein coupled receptor is associated with trimeric (alpha, beta, gamma) G-protein. binding of G protein recpetor ligand triggers intrnsic GTPase activity of G protein (GTP-->GDP+Pi)

2. Then the G protein alpha subunit causes the effector (in this case adenylate cyclaes) to cleave ATP and increase intracellular cAMP.

3. cAMP activates PKA by binding to regulatory subunit, and releasing catalytic subunit.

4. Catalytic subunit of protein kinase can phosphorylate proteins (which can either enhance or suppress protein activity). Phosphorus source is ATP

Front 8

G protein coupled receptors in the body:

Back 8

1. >1000 in the body
2. Adrenergic
3. Dopaminergic
4. Opioid
5. Sensory (rhodopsin, olfactory, taste)
6. Glycoproteins
7. Lipids and small molecules

Front 9

G protein subunits:

Back 9

1. 15 Alpha subunits known
2. 6 Beta subunits known
3. 12 Gamma subunits known

Front 10

Effectors in G-protein systems

Back 10

can include
1. adenylyl cyclase
2. phospholipases C and A2
3. cGMP phosphodiesterase
4. K+ channels
5. Ca++ channels

Front 11

Calcium levels must be regulated inside the cell. What are some cell functions that require regulation of calcium levels?

Back 11

1. Gene expression
2. Secretion
3. Enzyme activity
4. Ion transport
5. Neurotransmission
6. Cell adhesion
7. Cell division
8. Motility/cytoskeleton restructuring

Front 12

How are calcium levels regulated?

Back 12

A receptor operates through a G protein which acts on an effector system that can regulate intracellular Ca2+ levels. You want this to happen at a certain level and on a certain time. (chemoattractant signals often act through this pathway)

Front 13

Intracellular vs. Extracellular Calcium levels

Back 13

10^-6 M (micromolar) inside 10^-3 M (millimolar) outside

Front 14

IP3 binding to the ER allows calcium in the ER to be:

Back 14

A signal that promotes IP3 levels to increase, promotes IP3 binding to ER (which is Ca2+ rich) and allows Ca2+ entry into the cytosol.

Front 15

How can calcium (elevated) contribute to cell injury?

Back 15

1. ATPase becomes active, and ATP is lost
2. Phospholipases become active, and membrane damage to lysozomes, nucleus, and mitochondrion occurs
3. Proteases become active and wreck cytoskeleton and cell membranes
4. Endonucleases become active and break down chromatin
5. Mitochondrial permeability increases-->loss of membrane potential.

Front 16

Phosphoinositides:

Back 16

1. They are under the control of G protein coupled receptors. The alpha subunit acts on phospholipase C.

2. Phospholipase C then cleaves PIP2 from the plasma membrane (which is released as IP-3).

3. IP-3 binds to the IP-3 R onthe ER (where calcium stores are located), which triggers calcium entry into the cytosol.

4. Calcium can then bind to calmodulin, which mediates calcium-dependent activities inside the cell including effects on: ENZYMES, ION CHANNELS, TRANSCRIPTION
FACTORS, CYTOSKELETAL
PROTEINS, and RECEPTORS

Front 17

Calmodulin

Back 17

A calcium binding protein that can interact with other enzymes and channels inside the cell and can effect their activity.

Phosphorylase kinase (involved in glycogen metabolism)--one of its' subunits is calmodulin. So calcium modulation in muscle cell can activate phosphorylase kinase enzyme via Ca2+/calmodulin.

Front 18

Following signaling to upregulate behind, what is involved in a second phase of signal transduction?

Back 18

IP3 that is cleaved leaves diacylglycerol behind, which allows for docking of protein Kinase C, which then becomes active and induces protein phosphorylation events that induce some form of cellular effect.

So some signal has to first signal IP3 levels to release by releasing membrane bound IP2 via phospholipase C. Then protein kinase C can bind to the remaining diacylglycerol-->cellular effect.

Front 19

Effector enzyme of IP3 pathway

Back 19

phospholipase C

Front 20

Receptor tyrosine kinase

Back 20

1. Receptor tyrosine kinase have a ligand binding domain, a membrane-spanning domain, and a cytoplasmic domain with intrinsic kinase activity.

2. The cytoplasmic domain can bind ATP, and add phosphate to tyrosine (Y or Tyr) residues in the cytoplasmic domain.

3. An adapter protein can then bind to the phosphorylated tyrosine residues-->response..

Front 21

What on the adaptor protein allows the recognition of tyrosine phosphate patterns?

Back 21

SH2 domain (named so from the original discovery).

If something mutates SH2 domain, then that's the end of your response, and you can't carry out the physiological response.

The adapter protein has regions that know when the ligand is out there and when the information is trying to be transmitted throughout the cell.

You have to do it through the structural domains of the adapter proteins.

Front 22

Following ligand binding to receptor tyrosine kinase, what are key links in initiating characteristic cytokine /growth factor signal cascades?

Back 22

Following receptor dimerization and autophosphorylation, SH2 and docking are key links.

Front 23

What can growth factor receptors do that other receptor systems cannot?

Back 23

Dimerize

Front 24

SH2

Back 24

The src homology domain. SH2 is a sequence of amino acids found in certain proteins. SH2 allows these proteins to recognize and bind phosphorylated tyrosine residues in the cytoplasmic domain of a growth factor receptor.

Front 25

JAK/STAT Pathway

Back 25

Figure 1. The JAK-STAT Pathway. JAK phosphorylates the cytoplasmic portions of cytokine receptors, which aggregate in response to ligand binding. STAT proteins are recruited to the resulting binding sites. The STATs are subsequently phosphorylated (by JAK), creating dimers capable of accumulating in the nucleus, binding to promoters, and activating transcription through recruitment of the RNA polymerase II machinery. The signal-transduction cycle is terminated by dephosphorylation of activated STAT proteins by nuclear phosphatases.

Mutant STAT proteins unable to bind DNA can nonetheless interact with wild-type STAT proteins, resulting in nonproductive dimers that have a dominant negative effect on STAT function.

Front 26

MAP (Mitogen Activated Pathway)

Back 26

1. Growth factor binds to the EGF receptor.
2. After several phosphorylation steps, MAP kinase becomes active, where it enters the nucleus (where it can activate transcription factors via phosphorylation)

3. MAPK phosphorylates a TF for MYC which binds to the MYC promoter.

4. MYC then acts as a TF for early cyclin and CDK genes.

5. CDKs exist throughout the cell cycle, and cyclins are either S (for "s" phase) or M (for "m phase). When M/S cyclins join with CDK, they enter into the M or S respectively, and upon leaving the S or M phase, the cyclins dissociate from CDK and are degraded.

Front 27

S cyclins

Back 27

S cyclins bind to CDK and promote transition from G1-->S phase.

Transition from S-->G2 results in S cyclin dissociation and degradation.

Front 28

M-cyclins

Back 28

M-cyclins bind to CDK and promote entry from G2-->M phase.

Transition from M-->G1 results in M-cyclin dissociation and degradation.

Front 29

What are three defects in EGF receptors that that can lead to cancer?

Back 29

a. constitutive activation (ex: spontaneous dimerization of EGF receptors due to missing ligand binding domain-->ie glioma)
b. increased receptor number
c. increased ligand levels

Leads to more positive signal, and increased adapter molecule activity.

Front 30

In addition to changes in growth factor receptors, what mutations in growth control pathways can lead to human cancers?

Back 30

1. GTPase mutation means that GTP stays bound to RAS longer, leading to longer RAS activation.

2. Defective GAP binding means more active "ras"

3. RAS overexpression means that there is more "ras" that can be active at any given time.

In short, anything that activates "RAS" more can lead to cancer.

Front 31

Summarize the MAP kinase pathway, and active vs. inactive ras.

Back 31

1. Growth factor receptor binds growth factor leading to tyrosine kinase activity and phosphorylation of tyrosine residues.

2. adaptor proteins (in this case GEF/GAP) bind to cytoplasmic domain via SH2 homology.

3. RAS (gdp-bpound) then is exchanged for GTP, which activates RAS.

4. Ras can then activate MAP-KKK (RAF) -->MEK-P-->ERK-P (ERK-P Enters the nucleus, and phosphorylates the transcription factor which encodes for genes that control cell division, etc.

This makes it clear that activate RAS is actually what gets us into the chain of phosphorylation events that gets us into cell division, etc.

Front 32

What two proteins operate between the two forms of RAS?

Back 32

GAP (GTPase activity) and GEF (exchanges GTP for GDP)

Front 33

Retinoblastoma protein

Back 33

Remains bound to E2f. The cyclins/CDK Kinases produced from MYC (the map/kinase cascade) all the way when growth factor bound, phosphorylate RB, releasing E2F transcription factor which transcribes early genes needed for the G1-->S.

1. Thymidine Kinase
2. DHFR
3. DNA polymerase
4. Chromatin Proteins

Front 34

50% of all human cancers are due to:

Back 34

p53 mutations.

Front 35

What are 5 ways that p53 acts to suppress tumor activity?

Back 35

1. Induction of p21 which inhibits cyclin dependent kinase activity (stops division)
2. p21 binds to and inhibits DNA polymerase (stops DNA synthesis)
3. p53 induces GAPD45, a DNA repair protein (induces DNA repair)
4. p53 also binds ERCCC3 (an excision repair molecule that removes damaged DNA)
5. p53 induces apoptosis factors (BAX) to destroy damaged cells (triggers apoptosis of damaged cells)

Front 36

p21

Back 36

Inhibits cyclin dependent kinase activity

Front 37

BAX

Back 37

p53 induces BAX to destroy damaged cells

Front 38

GAPD45

Back 38

p53 induces GAPD45, a DNA repair protein

Front 39

ERCCC3

Back 39

p53 binds ERCCC3, an excision repair molecule (facilitates removal of damaged DNA)

Front 40

p53 signaling

Back 40

A DNA mutation may trigger phosphorylation of p53 (which is bound up by MDM2. Active, phosphorylated p53 then can bind p21 (a CDK inhibitor protein) which can make the S-cyclin/CDK complex in an inactive state (also has other effects like activation of BAX etc.)

Front 41

ATP levels in the cell are controlled by what two mechanisms?

Back 41

1. Substrate level phosphorylation (adding a phosphoryl group to ADP)

2. Oxidative phosphorylation

2. Oxidative phosphorylation

Front 42

How do nutrients from food become electrons that enter oxidative phosphorylation cycle?

Back 42

Nutrients from food (glucose) are broken down into CO2 water in small oxidative stpes in cells.

This doesn't occur directly, since the glucose breakdown actually reduces NAD+ to NADH, which serves as a shuttle that carries electrons to NADH dehydrogenase (Complex I) of the mitochondrial ox. phos system.

Front 43

How does electron flow accomplish cellular work?

Back 43

The electron flow produced from glucose breakdown and the corresponding NAD+-->NADH reduction generates ATP which accomplishes cellular work.

Front 44

What happens in between the oxidation of NADH and the reduction of O2 such that this energy can be used to synthesize ATP?

Back 44

Ox. phos creates a proton motive force and Pi and catalyzes ATP synthesis.

This is coupled to an enzyme mechanism that binds ADP.

Front 45

Oxidative Phosphorylation System

Back 45

Complex I/II- NADH carries electrons to complex I, which are transferred to FMN and Fe-S and finally from Q-->QH2.

Complex III- QH2 (ubiquinol)electons are removed and passed to cytochrome 1 (now in the reduced form)

Complex IV- Reduced cytochrome c moves from complex III, docks with cytochrome oxidase (complex IV) and transfers electrons to complex IV for O2 reduction to H2O.

Front 46

Summarize the reactions of oxidative phosphorylation and summarize which compartment of the cell they occur in.

Back 46

Complex I/II- NADH-->NAD+ + QH2

Complex III: QH2 + 2 cyt c1 (oxidized) -->2 cyt c1 (reduced) + 1Q

Complex IV: 4e- (from cyt c1 reduced) + 4H+ + O2--> 2H2o

O2+ 2 (NADH + H+) --> 2NAD+ + 2H2O

Front 47

Components of the proton motive force in oxidative phosphorylation that drives ATP synthesis include:

Back 47

1. a membrane potential (differences in charge).

2. differences in pH (proton gradient)

Front 48

ATP Synthase alpha subunit

Back 48

contains 2 partial channels, in contact w/ different sides of the membrane.

H+s moves through one of the partial channels to the center of the membrane, binds to one of the 10 c subunits (Asp-61) and is then carried to the other partial channel by rotation of the subunit complex

Front 49

ATP snthase b and delta subunits

Back 49

anchors a to the alpha3beta3 hexamer.

This makes it so that rotation of c relative to "a" will drive rotation of gamma relative to the alpha 3 beta 3 hexamer.

Front 50

Gamma subunit of the ATP synthase

Back 50

The asymetric gamma subunit rotates 120 degrees counter clockwise, driving conformaional changes in the 3 catalytic sites that alter their affinities for substrate and product.

The conformations are TLO (Tight, loose and open).

One 120 degree displacement of gamma makes 1 ATP. (One full turn yields 2.5 ATP).

Front 51

Explain how the gamma subunit rotation drives conformational changes that promote ATP synthesis?

Back 51

Part of the beta subunit undergoes conformational changes as a result of gamma subunit rotation.

1 120 degree displacement makes 1 ATP, whereas one full turn makes 2.5 ATP.

The tight site (T) opens (O)--releasing ATP. Then the empty O subunit allows association of ADP and Pi.

Front 52

SGLT-1 Transporter

Back 52

A glucose symporter that requires sodium to be co-transported from the lumen into the intestinal mucosal cell.

As a result of sodium levels being pumped into the intestinal mucosal, a Na+/K+ ATP aes must pump potassium in and sodium out of the mucosal cell.

Front 53

GLUT 2 Transporter

Back 53

Allows direct exit of glucose from the intestinal mucosal cell into the blood.

Front 54

Ion channels function to:

Back 54

1. Osmotic stability in all cells
2. Allow neurons to signal
3. K+ 10,000 times more permeant than Na+ (even though it's larger)

Front 55

How do ion channels function?

Back 55

Ion channels have a large fluid-filled vestibule on their cytosolic face. Hydration of ions such as K+ allow for transport of ions.

The hydrated ion then moves toward the channel, which has a backbone of carbonyl oxygens. There are alternating K+ binding sites within the channel as well. A helix dipole within the channel inter-membrane proteins provide stabilization of K+.

Anions move through different channels with a charged face (inside of pore) and hydrophobic face (adjacent to membrane)

Front 56

Mitochondrial disease characteristics:

Back 56

1. 10-15/100,000 births
2. lactic acidosis common
3. Maternal inheritance

Front 57

Mitochondrial genome

Back 57

a. Inherited maternally
b. 2-10 per mitochondrion
c. mutations 10x more frequent than in nuclear DNA
d. no introns
f. encodes: 13 protein subunits of ox phos system, 22 transfer RNAs, and 2 ribosomal RNAs

Front 58

Superoxide dismutase

Back 58

catalyzes formation of dihydrogen peroxide from free oxygen radicals.

Front 59

Catalase

Back 59

During episodes of oxidative stress, reduced (unbridged) sulfur groups rae are oxidzed and link to form a protein thiol bridge.

This oxidation is linked to catalase activity that breaks hydrogen peroxide down into H2O.

Front 60

The fenton reaction

Back 60

This is an iron-salt dependent decomposition of dihydrogen peroxide, geerating the highly reactive hydroxyl radical, possibly via oxoiron.

In the fenton reaction Fe2+ (ferrous) is oxidized to Fe3+.

Front 61

Catalase activity is linked to what other system?

Back 61

Glutathione peroxidase. (involved in formation of a protein thiol bridge.)

Subsequent activity of glutathione reductase reduces and breaks the protein thiol bridge so that another round of catalase can hop in and break down some more dihydrogen peroxide.

Front 62

What are some cellular effects of oxygen toxicity (radicals)?

Back 62

1. Lipid peroxidatoin
2. Protein damage
3. DNA damage
4. Cell injury

Front 63

What are some pathological consequences of defects in superoxide dismutase?

Back 63

1. Death 8 days post-natal due to dilated cardiomyopathy
2. Biochemical defects
3. Complex II reduced 60-80% in heart/skeletal muscle
4. Complex I reduced 41% in heart
5. 89% reduction in aconitase
6. Result-->electron transport and TCA cycle down.

Front 64

Superoxide dismutase

Back 64

formation of dihydrogen peroxide from free oxygen species.

Front 65

What is the end result of defects in superoxide dismutase?

Back 65

Downregulation of electron transport and TCA cycle.

Front 66

How do SOD defects reduce aconitase, complex I and complex II?

Back 66

The superoxide anion elevation due to SOD damages Fe-S centers in complex I and II.

Aconitase is also damaged in the TCA cycle.

Front 67

Events in apoptosis:

Back 67

1. "Protein cleavage" --> Activation of caspase enzymes (cysteine proteases) which hydrolyze nuclear and cytoskeletal proteins.

2. "Protein cross-linking" --> Cytoplasmic proteins are covalently linked to facilitate shrinkage.

3. "DNA fragmentation" --> Activation of nuclear endonucleases that cleave DNA.

4. "Phagocytosis" --> Phosphatidyl "serine" and "thrombospondin" on the apoptic bodies allow recognition and engulfment by macrophages.

Front 68

During apoptosis, what enzymes facilitate protein cleavage?

Back 68

"Caspase enzymes" (cysteine proteases) which hydrolyze nuclear and cytoskeletal proteins.

Front 69

During apoptosis, what happens to cytoplasmic proteins?

Back 69

They are linked covalently to facilitate shrinkage.

Front 70

What happens to DNA during apoptosis?

Back 70

Nuclear endonucleases that cleave DNA are activated, and this breaks up some DNA.

Front 71

What happens during to the cell following apoptosis?

Back 71

Phosphatidyl serine and thrombospondin on the apoptic bodies allow recognition and engulfment by macrophages.

Front 72

What are the cellular events that promote apoptosis?

Back 72

Apoptotic stimuli cause the mitochondrion to release cytochrome c-->binding of cyt c to apf-1.

ATP then supplies energy to form "the apoptosome" (which is apaf-1/cyt c aggregatation.) Procaspase 9 then binds to the apoptosome, which leads to activation of procaspase 9.

Active procaspase 9 (which is procaspase-3,7--> active caspase 3,7) leads to activation of the caspase cascade, and effects BCL-XL, and BCL-2 etc./triggering events that lead to apoptosis.

Front 73

When mitochondria are exposed to ROS a nonspecific inner membrane channel is formed. This channel is known as the:

Back 73

Mitochondrial transition pore.

The mitochondrial transition pore (MPT) is composed of several proteins including:
1. Porin
2. ANT
3. Bax
4. Bcl2
5. cyclophilin D
6. the benzodiazepine receptor

Front 74

Components and function of the mitochondrial transition pore.

Back 74

1. Porin
2. ANT
3. Bax
4. Bcl2
5. cyclophilin D
6. the benzodiazepine receptor

Opening of the mitochondrial transition pore leads to membrane potential collapse and mitochondrial swelling.

Front 75

Following opening of the MPT, what happens to the mitochondria?

Back 75

The swelling and distortion of the mito membrane structure permits loosely associated cytochrome c and other factors to escape the mito and enter the cytosol of the cell.

Released cyt c serve its apoptotic function by interacting with Apaf-1,forming an apoptosome and activating the CASPASE CASCADE.

Front 76

Factors that can promote formation of the mitochondrial transition pore (MPT):

Back 76

Kindly note that the MPT can be opened by (1) an increase in Calcium ion concentration in the mito, (2) increased ROS exposure as just described and (3) decline in energetic capacity and a loss of membrane potential.

Front 77

Cytochrome p450

Back 77

A drug "x" + O2 + NADPH + H+ --> XOH + H2O + NADP+

The way this rx. system works is that NADPH enters the NADPH cyp450 reductase. This leads to FAD-->FMN, and the shuttling of electrons to the cyt p450 oxidase.

The cytochrome p450 has iron centers which allow drugs to be oxidized via hydroxylation. Water is formed and NADP+ remains.