Download MCB 2050 Midterm Questions With Complete Solution5. and more Exams Nursing in PDF only on Docsity!
MCB 2050 Midterm Questions With Complete Solutions What is the active G-protein able to bind to? Downstream effector proteins What is one of the most frequently activated enzymes in signal transduction? Adenylyl cyclase What does adenylyl cyclase do? Convert ATP to cAMP What is the structure of GPCRs? 7 transmembrane alpha helices, with N terminus projecting out and C terminus projecting in What makes the ligand binding domain of a GPCR? The combination of the exterior parts of the protein (links between helices) What is the G-protein structure? Galpha = GTPase switch protein. Attached to GTP/GDP, hydrophobic end keeps it anchored to membrane Gbeta/gamma = rest of it, Ga dissociates from it when active. Also bound to membrane What is the mechanism of G-protein switches?
- Ligand binds to inactive receptor, causing a conformation change
- Active receptor binds Galpha (bound to Gbeta and gamma
- Active receptor does GEF act - GDP leaves and GTP binds
- Active Galpha dissociates
- Galpha activates effector
- Galpha does GTPase to go back to inactive, turning off effector What is the FRET technique? Used to determine protein-protein interactions, one is tagges with CFP (cyan) and one with YFP (yellow). When excited and close, the light from cyan excites the yellow and yellow light is emitted. When far, only cyan is emitted. In graph: when light goes down, means Galpha dissociated, so the thing added was a ligand to the receptor What are the steps of the adenylyl cyclase pathway?
- ligand binds receptor
- receptor activates G protein
- G protein activates adenylyl cyclase
- Adenylyl cyclase makes cAMP The same pathway on a diff receptor can inhibit adenylyl cyclase
- cAMP activates PKA by binding to it's inhibitor
- inhibitor falls off, PKA goes into nucleus
- PKA phosphorylates CREB
- Ligand binds RTKs: dimerize and autophosphorylate
- SH2 in Grb2 recognizes the phosphorylated tyrosines and binds. SH3 of Grb2 recognizes prolines in Sos and bind
- Binding of Grb2 to RTK and Sos couples the receptor to inactive Ras (GDP)
- Sos acts as GEF and activates Ras
- active Ras-GTP detaches from Sos, but is bound to membrane
- Active Ras-GTP binds to inactive MapKKK (MapKKK is bound to 14-3-3, which inhibits it)
- Ras does GTPase, phosphorylating MapKKK, releasing 14-3- 3
- MapKKK phosphorylates MapKK
- MapKK phosphorylates MapK
- MapK activates p90^RSKinase
- Both MapK and p90Rskinase move to nucleus
- MapK phosphorylates TCF on promoter, p90rsk phosphorylates SRF
- 2 SRFs bind to promoter with TCF = mini enhancer, stimulates transcription What does PI-3 kinase do? It makes the signaling complexes (membrane bound 2nd messengers) that activate PKB: IP2 : Phosphoinositol 3,4-biphosphate or 4,5-biphosphate IP3: phosphoinositol 3,4,5-triphosphate What is the PI-3 kinase pathway?
- RTK activate PI-3 kinase, which is bound to them with SH
- PI-3 makes PI-3 phosphates IP2 and IP
- IP2 and IP3 (membrane bound) binds to PH domain of PKB (inactive PKB to partially active PKB)
- Once PDK1 and PDK2 phosphorylate PKB, it is fully active
- PKB makes cell survive by inactivating pro-apoptotic protein synthesis What are the master regulators of the cell cycle? Cyclin Dependent Kinases (CDKs) What are cyclins? The regulatory component of CDKs What are some regulatory mechanisms of the cell cycle?
- synthesis and degradation of CDKs
- inhibitory and activating phosphorylation of CDKs
- inhibitors Can a cell back down past G1? No. Comited, will die if it doesn't complete cell cycle What are positive regulators of the cell cycle? Cyclins: they set up transition to next stage and then get degraded What are negative regulators of the cell cycle? Checkpoints: will hold cell cycle if something is wrong What happens during G1? How many copies?
How are human cells undergoing mitosis studied for the cell cycle? The cells are flat during interface but round during mitosis What are tools to study the cell cycle? Cell synchrony (uses chemicals to arrest cell cycle and then wash away so all cells progress at same speed Flow cytometry (stain cells and can tell amount of DNA through laser reading) Where do mating pheromones arrest cells? Is it yeast or mammalian? G1, yeast Where does nocodazole arrest cells? Is it yeast or mammalian? Pro-metaphase, both Where do hydroxyurea or thymidine arrest cells? Is it yeast or mammalian? Early S phase, mammalian What is the difference between wee mutants and cdc mutants? Wee mutants do mitosis at a smaller size and have advanced progress through cell cycle Cdc mutants do mitosis at a larger size and have delayed progress through cell cycle What cyclins are during early G no cyclins
What cyclins are during mid G1 to end of G1? G1 cyclins and then G1/S cyclins. Prepares cell for S phase and starts transition, respectively What cyclins are right before S phase? SCF ubiquitin protein ligase: induses S phase by ubiquitinizing the inhibitor of S phase cyclins What cyclins are during S phase? S phase cyclins What cyclins are in Late G2? Mitotic CDKs - trigger mitosis What cyclins act before anaphase? APC/C ubiquitin protein-ligase: degrade securin Also degrades mitotic CDKs What are the three types of cyclin regulators? Kinases & Phosphatases Inhibitory proteins Ubiquitin protein ligases What do Kinases and phosphatases do Phosphorylate or dephosphorylate CDKs to activate or inhibit them What do inhibitory proteins do? Control activity of CDKs by binding to them - inhibiting
- CAK (CDK Activating Protein)
- Wee1 Kinase
- cdc25 phosphatase What does CAK do to regulate CDKs? It mediates phosphorylation to activate CDKs What does Wee1 kinase do to regulate CDKs? It phosphorylates mitotic CDKs to inhibit mitotic kinases in S- phase What does cdc25 phosphatase do to regulate CDKs? It removes the inbibitory phosphorylation of wee1 on mitotic CDKs to activate mitotic CDKS Also works on G1/S How does the G1/S phase transition work in yeast at the level of CDK expression?
- Whi5 inhibits SBF on promoter of cyclin 1 or 2 CDK
- Nutrients stimulate the production of cyclin 3 CDK, which will phosphorylate Whi5, which dissociates from SBF
- SBF can now act as a txn activator for cyclin 1/2 CDK
- when cyclin 1/2 CDK are translated, they will initiate cell division (budding, S phase and spindle formation) *cyclin 1/2 CDK phosphorylate Whi5 too How does the G1/S phase transition work in metazoans at the level of CDK expression? 6 steps
-Rb binds and inhibits E2F on the promoter of Cyclin E/A
- Growth factors promote the transcription of Cyclin D, which will phosphorylate Rb (gets released from E2F)
- E2F can now act as txn activator for Cyclin E/A, which gets transcribed
- Cyclin E/A are G1/S phase cyclins and will initiate S phase
- Cyclin E/A phosphorylate Rb as well, creating a +ve feedback loop
- Cyclin E/A support cell through G1/S phase, through restriction point and initiation of DNA replication What is the process of the proteolyis of S phase Inhibitor Sic 1?
- Sic 1 is bound to S phase CDK, inhibiting it
- G1/S cyclins assemble in late G1 and phosphorylate Sic
- SCF ubiquitin protein ligase ubiquitinylates Sic
- Sic1 gets degraded
- Now S phase CDK is free to trigger DNA replication by phosphorylated MCM helicase What would happen if only 1 phosphorylation of Sic1 was needed? Think of graph Sic 1 levels would slowly get phosphorylated as the levels of G1/S cyclin CDKs rise slowly, so the transition G1-S would be long What actually happens with the phosphorylation of Sic1?
- event sensors
- signalling pathway
- effector, which halts cell cycle/activates repair What happens at G1/S checkpoint with TOR in yeast vs mammals? In yeast: TOR regulates activity of ribosomes to make proteins for cell division In mammals: TOR regulates activity of CDKs and synthesis of genes What is the DNA damage response system chain? Damaged DNA activates ATM and ATR ATM and ATR activate chk1 and chk2 protein kinases chk1 and chk2: -induce DNA repair
- activate tumor suppressor txn factor p53:
- activates txn of p21 to arrest cell cycle
- activates apoptosis through Bax, PUMA and Noxa
- inhibits cdc25 (cdc25 activates G1/S phase CDKs) What is a G1 cyclin? What does it act on? Cyclin D (acts on Rb to start G1/S phase cyclin production) What are G1/S phase cyclins? What do they do?
Cyclin E and A, inactivates cdh1 and phosphorylate S phase inhibitor What do S phase cyclins work on? Pre-replication complex What is apoptosis? Cell death through regulated release of caspase proteases What is the difference between necrosis and apoptosis? Necrosis = cell rupture, contents out and triggers inflammatory response Apotosis = cell death but contents are packaged in apoptotic bodies What are the steps of apoptosis? -chromatin condensation -cell shrinkage -parts of cell membrane break off (fragmentation, blebbing) -Phagocytosis What happens to all DNA in an apoptotic cell? All chromatin How does DNA fragmentation occur? Linker DNA between nucleosomes is cleaved by nuclease - makes less than 2N DNA How can we detect apoptosis?
What are the BH domains? Which ones are on pro survival and pro apoptotic proteins? Which proteins have the BH domains BH are Bcl2 homology domains. They bind to Bcl2. Pro survival proteins (Bcl2) = BH4 3 1 2 transmembrane Pro apoptotic proteins (Bak, Bax...) = BH3 1 2 transmembrane What are BH3 only proteins? Only have BH3 and a hydrophobic domain They regulate pro-survival proteins Bad, Bim, Puma What is the intrinsic death pathway?
- BH3 only proteins Bad, Bim and Puma are released
- They bind Bcl-2 and make it release Bak/Bax
- Bak/Bax form a channel on Outer Mitochondrial Membrane
- Cyt c leaves mitochondira IMM
- Cyt c binds Apaf-1 and activates it
- Apaf 1 converts inactive caspase 9, the INITIATOR caspase to an active initiator caspase 9
- initiator caspase 9 turns procaspase 3 into the EXECUTIONER caspase 3, which cleaves proteins and lead to cell death What stimulates the production of Bad? Trophic factor abscence (usually, trophic factor is inactive = constantly telling cell to live by inhibiting Bad)
What stimulates the production of Bim? Disruption of integrin signalling (cell isn't attached to other cells) What stimulates the production of Puma? DNA damage - Puma is synthesized in nucleus Does the extrinsic death pathway go through the mitochondria? No. Directly activates procaspase 8 What are the 3 steps of cancer progression?
- Mutations and genome destabilization
- Evasion of cancer cell elimination
- Tumor growth and dispersal (metastasis) Mutations on what cause genome destabilization? P Inhibitors of CDKs ATM/ATR DNA repair genes Mutations in what stops growth control? Cell cycle regulatoory proteins: -G1/S cyclins -Regulators of G1/S like Rb
- Growth factors receptors and signal transduction proteins Mutations in what block apoptosis pathway? Bcl2, Bim, Apaf
Cell looses ability to repair DNA, so mutations occur at a higher rate Deregulation of cell growth and cell death pathway occur due to what?
- hyperactivation of cell growth signalling
- loss of negative growth signalling Are signal receptors, signal transduction proteins and txn factors potential oncogenes or tumor suppressors? Potential oncogenes What are cell cycle control proteins, DNA repair proteins are negative regulators of signal transduction? Tumor suppressors Are apoptotic proteins tumor suppressors or protooncogenes? Both: pro-apoptosis = tumor suppressors, pro-survival = protooncogenes What is the progress of colon cancer?
- Polyp = loss of function in APC, a tumor suppressor gene - grows into tumor
- Adenoma cancer = gain of function in Ras (oncogene), now always stays on GTP so always active so growth signals always on
- Malignant carcinoma = loss of function in p53 - no apoptosis or stops in cell cycle
What is the HER2 receptor mutation? How does it lead to cancer? HER2 = RTK Normally, valine on transmembrane domain (hydrophobic) Mutated to a glutamine, hydrophilic, which will attract the other HER2 and dimerize Result: always active, ligand-independent oncogene What is the EGF receptor mutation? How does it lead to cancer? EGF = RTK receptor A deletion makes it oncogene by getting rid of exoplasmic domain: can't bind ligand anymore, always on, ligand independent What are the protooncogenes in the RasMAP pathway? RTK, Grb2, Sos, Ras, MapKKK, MapKK, MapK, txn factors (TCF and SRF) What is the tumor supressor in RasMAp pathway? NF1 (negative factor) What does normal stimulation of fos and myc genes look like? Both are transcribed after growth serum happens, and degraded after a bit fos and myc are txn factors What does abnormal stimulation of Fos look like? fos mutation makes it oncogene, and it's mRNA stays abnormally stable (growth for longer than usual)
