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72 Cards in this Set
- Front
- Back
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Three methods cells use to internalize extracellular materials |
1. Phagocytosis - macrophages, uncommon 2. Pinocytosis - make small vesicles from plasma membrane and bring liquid into the cell 3. Receptor-mediated endocytosis - receptor, vesicle, endosomes |
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Low-density lipoprotein (LDL) |
Soluble complex that transports lipids and cholesterol
- Amphipathic shell: single layer of phospholipids and unesterified cholesterol on the outside, with apolipoprotein B band - Apolar core - mostly cholesteryl esters |
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Clathrin/AP-coated vesicles
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Vesicle has two layer coat: clathrin and adapter protein complexes (AP2 for PM) - AP complexes recognize sorting signal of receptor, start budding - dynamin forms rings around the vesicle neck, polymerizes, GTP hydrolysis to pinch off vesicle NPXY motif on cytosolic side of receptor recognizes AP2 proteins AP2 proteins and clathrin fall off via GTP hydrolysis after internalization, then called early endosome |
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pH-dependent binding of LDL particles to the LDL receptor (LDLR) |
Three LDLR domains: - short C-terminal cytosolic segment with NPXY sorting signal motif - N-terminal exoplasmic segment with ligand binding domain and b-propeller domain - Ligand binding arm made of 7 cysteine rich repeats binds to Apolipoprotein B of LDL at pH 7 (cell surface) - NPXY (Asn-Pro-X-Tyr) domain recognized by AP2 complex, starts formation of clathrin vesicle - in late endosome, pH 5 -> histidine residues in b-propeller domain are protonated, then bind with high affinity to negatively charged residues in ligand binding arm, causing arm to move and release the LDL particle |
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Possible reasons why you wouldn't internalize LDL? What happens when you don't internalize LDL? |
Possible reason: - No LDL receptor - receptor binds LDL poorly - receptor can't internalize LDL (problems with AP2 etc.) What happens: - end up with lots of fat and cholesterol in bloodstream with nowhere to go - LDL starts precipitating out and making plaque in bloodstream in arteries - heart attack and stroke before late 20s |
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Acidification of endosomes and lysosomes |
Endosomes and lysosomes have V-class proton pumps that transport H+ into endosomes via ATP-dependent mechanism At the same time chloride channel allows chloride ions to flow into vesicles, anions passively follow the protons You end up with high concentrations of H+ and Cl- in the vesicle -> low pH in lumen |
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Endocytic pathway for internalizing LDL |
1. At cell surface, neutral pH, LDL receptor binds to ApoB 2. NPXY motif binds to AP2, clathrin recruited 3. Dynamin and GTP hydrolysis makes vesicles bud off 4. GTP hydrolysis makes coat proteins fall off -> early endosome, pH starts dropping 5. Early endosome fuses with late endosome 6. Acidic pH in late endosome - protonation of histidines in b-propeller causes release of LDL and receptor binds to itself 7. Late endosome fuses with the lysosome and LDL particles are broken down Meanwhile, LDL receptor recycles to cell surface, neutral pH then causes receptor to dissociate from itself to be ready to bind to new LDL |
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Transferrin cycle |
Used to get iron (a cofactor for a lot of enzymes) into the cell even though highly charged 1. Protein (homodimer) called apotransferrin bidns to Fe3+ -> ferrotransferrin 2. Ferrotransferrin binds to its receptor 3. Receptor recognized by AP2, clathrin pit, Dynamin + GTP causes vesicle to bud off 4. AP2 and clathrin fall off the vesicle, early endosome, starts to acidify 5. Fuses with late endosome, pH 5 - Fe3+ released, apotransferrin stays bound to receptor 6. Fe3+ reduced to Fe2+, exits late endosome with protein help 7. Apotransferrin and receptor still bound are recycled to the cell surface, neutral pH causes the apotransferrin to dissociate from the receptor Ferrotransferrin binds to the receptor at neutral pH, Apotransferrin binds at low pH |
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Autophagy (self-eating) |
Lysosomal/vacuolar degradative pathway conserved in eukaryotic organisms - mediates turnover of long-lived proteins and excess or aberrant organelles, gives extra energy when starving, important in aging and disease (get rid of stuff not working) Lysosomal membranes and Atg proteins somehow form an autophagosome - single lipid bilayer Autophagosome fuses with lysosome, degrades everything (proteasomes?) |
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Atg 8 and Atg 5 |
Atg 8 and Atg 5 needed for formation of autophagosome - loss of Atg8 in fruit flies and loss of Atg 5 in mice -> aggregates of aberrant proteins in brain -> progressive neurodegeneration and early death - overexpression of Atg 8 in fly brains and Atg 5 in mice brains promotes lifespan extension |
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Apoptosis - vs. necrosis - purposes - general process |
Apoptosis is programmed cell death, Necrosis is where cells swell and burst because of a bad environment Purposes - ensures normal embryonic development - ensures proper tissue homeostasis in adult animals - keeps DNA and other stuff compartmentalized so it doesn't mess up other cells General Process: 1. Mild convolution, Chromatin compaction and margination, Condensation of cytoplasm 2. Breakup of nuclear envelope, Nuclear fragmentation, Blebbing, Cell fragmentation -> apoptotic bodies 3. Phagocytosis of apoptotic bodies |
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C. elegans |
Nematodes, Gave us majority of information about apoptosis Advantages: - small (1 mm) - transparent - every single cell mapped out (for both forms) - genome fully sequenced - many genetic mutants Has ced-3, ced-4, and ced-9 genes CED9 = Bcl2, Bax, Bak CED4 = Apaf-1 CED3 = Caspase 9 |
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Caspases |
Cysteine-dependent Aspartate-directed proteases (ex. CED3) Two types: 1. Initiator caspases - cleave inactive pro-forms of effector caspases, thereby activating them 2. Effector caspases - cleave other protein substrates within the cell to trigger apoptotic process Cleave pro-domain of initiator to activate caspase, cleaves other pro-caspases -> effector caspases Effector caspases cleave diff cell parts: - Inhibitor of DNase (ICAD) -> DNA fragmentation - Nuclear lamins -> fragmentation of nucleus - Cytoskeletal proteins Actin, myosin, alpha-actinin, tubulin, vimentin -> cytoskeletal disruption, cell fragmentation, membrane blebbing - Golgi matrix proteins -> fragmentation of Golgi |
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The Apoptosome |
1.4 megadalton wheel of death Made up of APAF-1, usually exists as monomer - cytochrome c binds to Apaf-1 in the cytosol (because of Bax channel) - Apaf-1 then forms heptomer apoptosome - apoptosome activates dimer of Caspase 9 (initiator) by binding to it*signal amplification - Caspase 9 activates effector caspases (like caspase 3) *signal amplification |
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Bcl-2 Family Proteins |
Prosurvival members: BH1-4, TM - Bcl-2 Pro-apoptotic members (form channels in outer mitochondrial membrane): BH1-3, TM - Bax, Bak BH3 only proteins (regulate Bcl-2 and Bax activity): BH3, TM Puma, Bim, Bad, Bid Bcl-2 originally isolated from B-cell lymphoma BH = Bcl-2 homology domain BH3 domain - shared domain, proteins bind - pro-survival and BH3 only proteins competing for spots |
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How DNA damage leads to apoptosis |
- Proteins scan DNA and look for damages - Damage found, triggers recruitment and activation of kinase ATM - ATM phosphorylates (and thus activates) p53 - p53 transcription factor increases expression of PUMA (BH3 only protein) - PUMA binds to and inhibits Bcl-2, which allows Bax/Bak to release cytochrome c from mitochondria - Apaf-1, apoptosome, caspase 9, caspase 3, death |
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Regulation of apoptosis by mitochondria |
On the outer mitochondrial membrane: Bcl2 interacting with Bax through BH3 domain - Bax monomers, no channel PUMA pathway (DNA damage) Bim: Disruption of integrin signalling -> rearrangement of cytoskeleton -> microtubules released Bim -> Bim binds to Bcl-2 -> Bax oligomerization |
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Intrinsic signals for apoptosis |
PUMA - DNA damage Bim - cytoskeleton rearrangement, integrin? Bad - loss of trophic factor All BH3 only proteins |
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Trophic factors and apoptosis |
Trophic factor: cells need it to survive, tells them to keep at it - normally trophic factor starts signalling cascade with PI-3 kinase and PKB to phosphorylate Bad (inhibiting it) and degrade it - loss of trophic factor -> active Bad accumulates in cytoplasm -> interferes with Bcl-2 (like PUMA) -> Bax oligomerizes |
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Extrinsic Apoptosis Pathway |
Cells receive signals from other cells to start apoptosis - Fas ligand on membrane of one cell binds to Fas receptor on another cell - Fas receptors come together and recruit FADD - FADD activates Caspase 8 (initiator) - Caspase 8 activates a bunch of other caspases including Caspase 3, AND cleaves Bid to BH3 only protein that inhibits Bcl-2 as well |
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Problems when apoptosis isn't regulated |
Too little - developmental defects - cancer - weakened immunity (autoimmune diseases) Too much apoptosis - Neurogenerative disorders (misfolded proteins in brain) - Alzheimer's, Parkinson's - Ischemia/reperfusion-associated injury - AIDS - Aging |
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Intracellular Second Messengers
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ATP + Adenylyl Cyclase -> cAMP - activates Protein Kinase A cGMP - activates Protein Kinase G and opens cation channels in rod cells IP3 - opens Ca2+ channels in the ER Ca2+ DAG - activates Protein Kinase C |
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G protein-coupled receptors - general structure - number of types - types of ligands - where they're found |
Seven transmembrane alpha helical regions (H1-H7) Four extracellular segments (E1-E4), E1 is N-terminus Four cytosolic segments (C1-C4), C4 is C-terminus C4 and C3 interact with trimeric G protein - 800-1000 types or more - ligands could be anything: light, neurotransmitters, hormones, pheromones, etc. - present everywhere, often targets of medication |
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General mechanism of activation of effector proteins associated with G protein-coupled receptors - Ga and Gg are tethered to plasma membrane via lipids |
1. Binding of hormone to receptor induces conformational change 2. Activated receptor binds to Ga subunit 3. Activated receptor causes conformational change in Ga subunit, triggering dissociation of GDP 4. Binding to GTP (abundant in cytosol) to Ga triggers dissociation of Ga from the receptor and from Gbg 5. Hormone dissociates from receptor, Ga binds to effector and activates it (sometimes Gbg) 6. Hydrolysis of GTP to GDP (sped up by RGS and GAP) causes Ga to dissociate from effector and reassociate with Gbg, returning system to resting state |
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Major classes of Mammalian Trimeric G proteins with their effectors |
Gas - Adenylyl cyclase, cAMP up - Ca2+ channel, Ca2+ up - Na+ channel, membrane potential down Gai - Adenylyl cyclase, cAMP down - Ca2+ channel, Ca2+ down - K+ channel, membrane potential up Gaq - Ca2+ channel, Ca2+ down - Phospholipase C, IP3 and DAG up Gat (rhodopsin in rod cells) - cGMP phosphodiesterase, cGMP down |
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Signal Amplification* - Fight or Flight Response (short term effects) |
- epinephrine released at low concentration - activates some adenylyl cyclase* - adenylyl cyclase makes cAMP* - 4 cAMP turns on PKA - PKA phosphorylates many different enzymes* - activates enzymes make a bunch of glucose* to make a bunch of ATP(*?) for processes Adrenaline rush! |
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Shutting off GPCRs |
After a certain amount of time, even if ligand is still bound, it'll shut off - receptor is phosphorylated on cytosolic side - Arrestin binds to phosphorylated sites so G protein can't bind, and also acts as adapter protein - recruits clathrin coated pits, endocytosis of receptor, amino acids of receptor recycled |
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Use of FRET to research GPCR pathway |
Use for drug tests, to see if artificial ligand has caused the G protein complex to dissociate - Attach CFP to Ga subunit and YFP to Gbg subunit - Use confocal microscopy and set laser to excitation wavelength of CFP (440 nm) - If the fluorescence detected is blue, Ga must be away from Gbg (Ga in active form) - If fluorescence is detected as yellow, Ga must be bound to Gbg (Ga inactive) |
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Regulation of Adenylyl Cyclase ("effector protein") |
Activation - stimulatory hormones: epinephrine, glucagon, ACTH - ligands bind to GPCR, Gas turns on Adenylyl cyclase - requires ATP to be cleaved to cAMP Inhibition - inhibitory hormones: PGE1, Adenosine - ligands bind to GPCR, Gai shuts off adenylyl cyclase - similar type of G protein complexes, just that one is stimulatory and one is inhibitory |
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Activation of PKA (by cAMP) |
PKA has 2 regulatory subunits (each with 2 cAMP binding sites, CNB-A and CNB-B) and 2 catalytic subunits - cAMP binds to the CNB-B sites, lowering the dissociation constant (Kd) - allows cAMP binds to CNB-A sites - once all four sites are filled, conformational change - catalytic subunit is released, activating PKA |
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Inappropriate activation of Adenylyl Cyclase - Vibrio Cholera (from dirty water) |
Vibrio cholera - produces toxin
Toxin enters enterocytes of small intestine using a GM1 ganglioside receptor - mimics ligand so its endocytosed into cell - toxin turns on Gas, ADP ribosylation (adds ADP and ribose, inactivates protein) causing Gas to always be on - Gas can't hydrolyze GTP to turn off, keeps adenylyl cyclase on, a lot of cAMP made - cAMP activates CFTR (chloride pump), chloride ions pumped out of cell, Na+ and water follows - causes a loss of water in the intestines -> diarrhea, dehydration - drink ions and sugar so that water comes back as well, also take antibiotics |
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Inappropriate activation of Adenylyl Cyclase - Bordetella pertussis (whooping cough) |
Pertussis toxin enters epithelial cells in the lungs - PTX ADP ribosylates ("inactivates protein") Gas, mass activation of Adenylyl cyclase - huge increase in cAMP levels, stimulates CFTR to pump out more chloride (and thus ions) from lung cells - mucous secretion and electrolyte/H2O accumulation in the lungs - causes whooping cough and could lead to secondary infection, treat with immunization |
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GPCR Activation of Transcription: CREB signalling - what is CREB - where is it located - function - requirements |
CREB = cAMP Response Element Binding Protein - located in the nucleus - helps recruit other DNA binding proteins to activate/repress hundreds of genes, binds thousands of promoters, lots of pathways - requires phosphorylation (kinases primarily phosphorylate serine, threonine and tyrosine) |
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CREB Pathway |
- G protein activation, increased adenylyl cyclase and cAMP levels, activates PKA - When PKA is activated (4 cAMP), catalytic domains enter the nucleus and phosphorylate CREB - p-CREB binds to promoter (in the cAMP response element = CRE) of a gene and recruits coactivators CBP or P300 - CBP and P300 recruit appropriate transcriptional machinery (CBP tells stem cell to stay undifferentiated and proliferate, P300 tells stem cell to differentiate) |
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CREB Pathway: Mutations of CBP
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CBP recruits transcriptional machinery to help stem cell stay undifferentiated, keep proliferating Mutations lead to Autosomal dominant disorders - mental retardation - polydactyly - Huntington's |
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Overview of Vision and the GPCR stuff |
Beta carotene (Vitamin A precursor) is oxidized to retinal Opsin is a GPCR! Rhodopsin = Opsin bound to retinal, found in disks in rod cells No light: rhodopsin with 11-cis retinal Light: retinal becomes all trans isomer, activates opsin to signal its Gat (transducin) After opsin dissociates, rhodopsin resets with 11-cis retinal again to shut off pathway, fast mechanism (evolution) |
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How is light perceived? More detailed pathway |
Rod cells in the dark have RMP of -30 mV because of a constant influx of Na+/K+/Ca2+ ions - cGMP present, it keeps gated cation channel open - Rod cells signal to the neurons and the brain perceives dark Photon of light causes retinal to separate from opsin - opsin Gat binds to GTP and dissociates - effector protein is a phosphodiesterase (PDE) that cleaves cGMP (opposite of cyclase) - cGMP levels fall, ion channels close, RMP drops (hyperpolarization) - less signaling to visual cortex, brain perceives light System resets when all trans retinal goes back to 11-cis, opsin rebinds to prepare for the next photon |
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Treating angina: vasodilation, relaxation of vascular smooth muscle |
- Acetylcholine ligand stimulates GPCR in blood vessel lumen - Gaq activates Phospholipase C - Phospholipase C cleaves PIP2 into IP3 and DAG (second messengers) (IP3 enters cytoplasm of endothelial cells, DAG stays in the membrane) - IP3 opens Ca2+ channels in the ER, Ca2+ enters cytosol - Ca2+ binds to Calmodulin - Calmodulin activates Nitric Oxide Synthase (NOS) - NOS converts Arginine and O2 into Citrulline and NO gas - NO gas diffuses into smooth muscle cells of blood vessel, activates Guanylyl cyclase (GTP into cGMP) - cGMP activates Protein Kinase G and causes vasodilation (relaxation of smooth muscle) -> blood pressure drops (Also amplification in this pathway) |
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How Viagra/Levitra works |
- Parasympathetic NS release NO in corpus cavernosum - NO receptors activate guanylyl cyclase, cGMP up and PKG activity up - causes vasodilation of smooth muscle resulting in increased blood flow - Viagra inhibits PDE5 that hydrolyzes cGMP to GMP, so vasodilation (and thus erection) is maintained Side effects: priapism, off target effects (other PDEs - heart attack, stroke, sudden hearing loss, cyanopsia) |
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Receptor Tyrosine Kinases (RTKs) - domains - possible ligands - general pathway |
Protein with three domains: extracellular (ligand-binding) domain, single transmembrane alpha helix, cytoplasmic domain with tyrosine kinase activity Ligands include growth factors and insulin Ligand binding, receptor dimerization, tyrosines phosphorylated, then need adapter proteins (ex. GTPase switch protein Ras) to bind to phosphates and transduce signal to downstream kinases - ultimately triggers changes in cell cycle and proliferation - aberrant signalling of RTKs at the root of many cancers |
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Activation of RTKs |
Ligand binds to monomer receptors in the membrane - facilitates dimerization (often homodimerization) of the receptors - Trans-autophosphorylation: each half of the cytosolic domain of the dimer phosphorylates each other on the activation lip tyrosines - Allows for phosphorylation of more tyrosines on cytosolic domains - phosphotyrosines serve as docking sites for adapter proteins with SH2 or PTB domains |
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RTKs: Adapter Proteins |
Contain unique domains that can recognize specific sequences, then eventually transmit the signal to Ras Common adapter domains: - SH2: src homology 2 domain - SH3 - PTB: phsophotyrosine binding domain, found on multidocking proteins, this is is a docking site for other signal transduction proteins - IRS-1: insulin receptor substrate protein, binds to PTB domain |
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Discovery of First oncogene (Src) |
Rous identified cancer-causing retrovirus (Rous Sarcoma Virus) - one of the genes in the virus encodes tyrosine kinase (src) involved in cell signalling - Src gene found in humans, highly conserved - cellular Src (c-Src) protein activity is regulated through phosphorylation, while v-Src has no inhibitory domain on the C-terminus to allow inhibitory phosphorylation - has SH2 and SH3 domains, Kinase domain |
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Ras |
GTPase switch protein, enzyme - Monomeric G protein - lipid-anchored to PM (like Ga subunit), but not directly linked to cell-surface receptors - Effector of RTK signalling - smaller than Ga - low GTPase activity - activated by adapter proteins (for one) Activity Regulation: Activator: GEF (guanine nucleotide exchange factor) Inactivator: GAP (GTPase accelerating protein) RasD is mutant constitutively active - no GTP hydrolysis - mutation of glycine-12 which prevents GAP binding - 20-30% of huamn cancers |
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EGF-induced Ras activation |
1. EGF binds EGFR, dimerization, trans-autophosphroylation sets up docking scaffold, GRB (growth factor receptor bound, adapter protein with no enzymatic activity) binds - GRB2 SH2 binds to phosphotyrosine 2. Assembly of multiprotein complex: two SH3 domains in GRB2 bind proline in SOS protein (GEF), SOS binds to Ras -> Ras now primed but still inactive 3. SOS (GEF) promotes dissociation of GDP from Ras, GTP binds, Ras-GTP active (dissociates from SOS) and triggers down stream kinase cascade with amplification: Ras -> Raf -> MEK -> ERK (or MAPK) MAPK = mitogen activated protein kinase ERK = extracellular signal-regulated kinase Pathway was discovered backwards, MAPK discovered first because so amplified |
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Ras/MAP kinase pathway |
RTK signalling -> Ras recruits, binds and activates Raf, GTP hydrolysis leads to dissociation of Raf from Ras -> Active Raf phosphorylates MEK -> MEK phosphorylates ERK Kinases: Raf, MEK, ERK Ras is a GTPase Raf = MAPKKK MEK = MAPKK ERK = MAPK Active ERK goes into nucleus and activates transcription factors for genes of cell cycle, cell growth, cell differentiation Raf, MEK, ERK all have off swtiches as well |
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Mutations in RTKs that promote proliferation in the absence of ligand - oncogenic receptors |
Mutation in transmembrane domain of RTK (HER2) can cause receptors to dimerize and autoactivate without ligand (Neu-neuroblastoma) Deletion in extracellular domain of EGF receptor - loss of ligand-binding domain can lead to constitutive activation of cytoplasmic kinase domain (ErbB oncoprotein) |
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Breast Cancer and HER family |
HER = human epidermal growth factor (EGF family) Four RTKs: HER1 to HER4, bind to EGF and related ligands HER2 doesn't bind ligand itself but can form heterodimers with the other HERs - results in activation with only one ligand instead of two High HER2 levels -> signalling cascades activated at much lower growth factor concentrations 25% of breast cancer patients have increased HER2 expression (over 100 fold)
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Herceptin |
Drug - monoclonal antibody
Binds to HER2 receptor and tags cell - the antibody can be recognized by immune cells - can also be recognized by other cell-surface proteins (receptor mediated endocytosis -> put receptor into lysosome) |
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Function of the Cell Cycle |
- mechanism by which life reproduces and passes on genetic information - ensures that DNA is replicated perfectly (but also organelles, enzymes, membranes, etc.) - segregate replicated chromosomes to 2 identical daughter cells - coordinate growth with division |
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Phases of the cell cycle |
Four phases: G1, S, G2, M G1 (Gap1) - generalized growth and metabolism - cells arrest to G0 when not dividing for a while - when very specialized like neurons or muscles - longest phase, variable in length (11 hours in mammalian cells) S (Synthesis) - DNA replication (6-8 hours) G2 (Gap2) - prep for mitosis (4 hours) M (Mitotic) and Cytokinesis (divides up cytoplasm) G1+S+G2 = interphase, cell growth, DNA open and being transcribed M - chromatin condensed with histones et al. Can use FACS and Hoechst Stain - stains DNA, amount of DNA shows what phase the cell is in Can see G2-M best in the microscope |
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Eukaryotic cell-cycle times |
Embryo - all cells dividing, no gap phases (S and M only because yolk stores have it all), 20-30 mins Yeast - single-celled and small, fast, 1.5-3 hours Skin cells, hair, intestinal epithelium - replaced often (intestinal about 12 hours) Mammalian fibroblasts in culture - 20 hours Human liver cells - 1 year, long interphase, maybe G0 Human nerve cells are terminally differentiated and mostly in G0 |
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M phase (mitosis) steps |
Prophase - nuclear envelope breaks down (mostly) - spindle apparatus forms Metaphase - chromosomes captured and aligned Anaphase - chromatids separate Telophase - reassembly of nuclear membranes Cytokinesis |
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S. cerevisiae cell cycle |
Budding yeast - cell cycle stage can be inferred by bud size - long G1 phase Entering S phase: forms bud that grows too - half of stuff goes into bud, smaller daughter cell G2 phase: nucleus entering bud M phase: when you see things condensing |
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S. pombe cell cycle |
Fission yeast, mother cell divides in half - hard to observe cell cycle stage except by yeast size - grows by elongation of ends G2 and M longer Cytokinesis - formation of septum |
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Yeast cells, cdc mutants |
Damage DNA - random mutation
Grow yeast cells and screen them for mutations in cell cycle - defects in specific proteins required to progress through the cell cycle cdc = cell division cycle mutants S. cerevisiae - cdc28 S. pombe - cdc2 |
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Cell cycle regulation - 3 protein families |
1. Kinases 2. Phosphatases (above two are enzymes) 3. Cyclins |
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Functional complementation |
Procedure for screening DNA library to identify WT gene that restores function of defective gene - plasmid with WT allele will complement the recessive mutation - find mutants and screen for temperature sensitivity (at what temp mutation manifests) - Buy plasmid with a random yeast gene and put it into the mutants to try to complement the mutation - keeping putting in random genes until you find the one that fixes the mutation - grow fixed colony, isolate plasmid, sequence the gene Discovery of CDC28 - the only CDK in S. cerevisiae |
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Control of G2 to M transition in S. pombe |
Gene for loss of cdc2 activity is recessive - cannot enter M phase - large cells Gene for gain of cdc2 activity is dominant - brings on M phase earlier - smaller cells Cdc2 (CDK) transcribed and translated throughout cell cycle, homologous to Cdc28 in S. cerevisiae - human CDKs can functionally complement these two (in the same gene?) ^proteins highly conserved |
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CDKs in Humans vs. Yeast, function of CDKs |
Humans have 4 CDKS, Yeast have 1 - CDKs only active in the stages of the cell cycle that they trigger |
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CDC2 activity - S. pombe |
Heterodimer of CDC2 (CDK) and CDC13 (mitotic cyclin) makes MPF (maturity promoting factor) Amount of Cdc2 is the same in all cell cycle changes Activity of Cdc2 (amount of cyclin) rises in G2-M transition |
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Cyclin Regulation of Cell Cycle (4 points) |
- they bind to and activate CDKs - they're only present during the cell cycle stage that they trigger and are absent otherwise (or low amounts) - divided into four classes based on presence and activity during cell cycle (G1, G1/S, S, M) - regulation via transcription and ubiquitin-mediated proteasome dependent degradation |
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Ubiquitin-mediated proteasome-dependent degradation (of cyclins in this case) |
Ubiquitin - small protein found everywhere in all euk cells - Polyubiquitination: Ubiquitin ligase adds ubiquitin molecules to a protein targeted for degradation (E3 recognizes target motifs, E2 adds) - once chain reaches a certain size, protein goes to 26S proteasome for degradation - ubiquitin molecules released and recycled |
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Regulation of MPF activity via phosphorylation of CDK (S. pombe) |
- As transcription and thus cyclin levels increase in the cell, it makes sure the CDK is inactive- Wee1 kinase phosphorylates Tyrosine 15 of CDK - at the right time, Cdc25 phosphatase removes the phosphate and activates the CDK (as G2 is completed) - CAK (CDK-activating kinase) also phosphorylates Threonine 161 to activate MPF Deficit of Cdc25 or Excess of Wee1 = large cells, not enough CDK activated, more G2 Excess of Cdc25 and Deficit of Wee1 = small cells, too much CDK, decreased G2 |
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What will the cell do with active MPF? |
Phosphorylation! - chromosome condensation - disassembly of nuclear envelope - interphase microtubule disassembly and mitotic spindle formation - Remodeling Golgi, ER (stops vesicular traffic) |
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Exit from mitosis - inactivation of MPF |
MPF should be inactivated at Anaphase - cyclins degraded through polyubiquitination - APC/C (Anaphase-Promoting Complex/Cyclosome) is the ubiquitin ligase - APC recognizes the destruction box (9 AA) on cyclins - During metaphase/anaphase the destruction box on the cyclins are exposed and can be identified by APC - low levels of cyclin, MPF drops and cell goes to Interphase |
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Overview of Cell cycle and different cyclin levels (humans) |
G1/S CDKs activate expression of S phase cyclin - SCF (ubiquitin ligase) and proteasome degrade G1 cyclins S phase CDKs, cyclins degraded by SCF/proteasome Cdc25 phosphatase/CAK activates mitotic CDKs -> MPF, activating early mitotic events Anaphase - phosphatases activate Cdh1 and APC ubiquitin ligases, proteasome degrades mitotic cyclins |
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Cell cycle checkpoints |
G1: damaged DNA G1-S: unfavorable extracellular environment S-G2: damaged or incompletely replicated DNA G2: damaged or incompletely replicated DNA M: chromosome improperly attached to mitotic spindle (metaphase) DNA damage checkpoints in G1, S, and G2 phases (more on this later) |
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DNA damage checkpoint |
DNA damage triggers protein kinase activation and phosphorylation of p53 (transcription factor, guardian of the genome) - p53 levels accumulate, activates transcription of p21 (CDK inhibitor protein CIP) - p21 inhibits cyclin-CDK complexes and halts cell cycle - p53 also increases PUMA, if enough PUMA, triggers apoptosis - if things aren't fixed fast enough |
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Review: the cyclin-dependent protein kinase system and how it's regulated |
CDK regulation - cyclin levels - kinases (inhibitory Wee1, activating CAK) - phosphatases (activating cdc25) - CDK inhibitors (CKIs), p21 Cyclin Regulation - transcription - ubiquitin ligases and degradation |
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Review: 5 major classes of cell-cycle regulatory proteins |
1. Cyclin-dependent kinases (CDKs) - serine/threonine kinases (Cdk1, cdc2, cdc28) 2. Cyclins - heterodimeric partners with CDKs, regulates activity 3. Cyclin-dependent kinase inhibitors - inhibit CDK activity by physically blocking activation or substrate/ATP access (CIP - p21) 4. Ubiquitin ligases - catalyse ubiquitination and degradation of cyclins - ex. APC/C, SCF 5. Transcription factors - promote transcription of cyclins, checkpoint genes - ex. p53 |
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Growth promoting and Growth inhibiting signals in the cell cycle |
Growth inhibiting signals - DNA damage - cell-cell contact - loss of mitogenic signal (ERK stimulating transcription?) - terminal differentiation - aging - CDK inhibitors - Ubiquitin ligases - Tumor suppressors (p53) Growth signals - mitogens (growth factors) - Transformation (viral or genetic alterations) - CDKs - cyclins - oncogenes |
