Cell bio notes 3/28-4/1
Cell bio notes 3/28-4/1 BIOL 30603
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This 14 page Class Notes was uploaded by Mallory Notetaker on Saturday April 2, 2016. The Class Notes belongs to BIOL 30603 at Texas Christian University taught by Dr. Akkaraju, Dr. Misamore, Dr. Chumley, Dr. McGillvray in Spring 2016. Since its upload, it has received 48 views. For similar materials see Molecular, Cellular, and Developmental Biology in Biology at Texas Christian University.
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Date Created: 04/02/16
SS Cell Bio Notes Exam 3 3/28/16 Continuing subject of G protein Targets: Enzymes Activated enzymes make second messenger signaling molecules What’s the first messenger? (ligand) Two major classes of enzymes: Adenylyl cyclase-> cAMP (second messenger) Phospholipase C-> inosital triphosphate & diacylglycerol (second messenger) Adenylyl Cyclase & cyclic AMP Usually the α subunit of G-protein switches on adenylyl cyclase which makes cAMP cAMP activates other enzymes, often protein kinase A (PKA) which in turn phosphorylates different target proteins -cAMP phosphodiesterase converts cylic AMP -> AMP cAMP Signaling Many cellular responses to extracellular signals are mediated by cAMP (you can make lots of cAMP AMPLIFYING SIGNAL HERE) -PKA is a kinase that adds a phosphate to a target protein -cAMP activates PKA Ex: adrenaline binds adrenergic receptor Consequences of this activation will vary depending on cell type Ex. Adrenaline acting on muscle cell -it resulted in glycogen break down because you will be needing lots of glucose for that muscle cAMP Signaling (another example) Different cell type Same extracellular signal Same extracellular receptor Same second messenger signal Same second messenger target Different PKA target -> transcription factor What will the cellular response be? -the outcome will be different -activating transcription, so cell will be producing proteins -changing protein synthesis Which response will be faster? This one or glycogen breakdown? -glycogen breakdown (because everything already made) -almost immediate (altering enzyme activity) there -here we are changing protein synthesis Cholera: Acute bacterial infection caused by ingestion of water contaminated with Vibrio cholerae. occurs when you don’t have access to clean water Sudden watery diarrhea and vomiting can result in severe dehydration. Left untreated, death may occur rapidly, especially in young children. Cholera Signaling B subunit binds to a receptor on the cell of the gut and then endocytosis and once in the cell the A subunit breaks free of B and goes and binds Gs-alpha which stimulates adenylate cyclase Cholera toxin (CT) is produced by the bacterium Vibrio cholerae (which stays in the lumen of intestine) -the reason you get such a strong response is because the CT modified a G- protein so it can’t hydrolyze GTP -can’t hydrolyze the GTP, so it is going to stay on (no way for protein to turn off) -constantly activating adenylate cyclase —> tons of cAMP -this causes Cl to leave the cell (lots of it) and this causes water to rush with it -you have lots of water rushing into the lumen of intestine (causees the extreme diarrhea -This is also how the V. Cholerae to spread because people have diarrhea and it gets in the water there (this is why watery diarrhea is beneficial to V. cholerae G protein Targets: Phospholipase C -another example but the beta gamma g protein activates phospholipase C -will cleave inositol phospholipid into two things (diacylglyercol and inositol 1,4,5 triphosphate) -inositol 1,4,5 triphosphate goes and opens Ca2+ channels in the endoplasmic reticulum -eventually Ca2+ and diacylglercerol will activate PKC Now Enzyme-coupled receptors Cytoplasmic domain acts as an enzyme or forms a complex with another protein that acts as an enzyme -causes dimerization -then autophosphorylation -once the dimers tails are phosphorylated they can interact with intracellular signaling pathways (phosphorylate signaling proteins) -they serve as a scaffold for lots of things to be stimulated at one time Largest class: Receptor tyrosine kinases (RTKs) (example) Tyrosine phosphorylation allows many signaling molecules to bind to cytosolic tails of receptor Simultaneously can signal along multiple pathways Different RTKs recruit different intracellular signaling proteins Two common ones PLC and Ras Ras Monomeric GTPases - just one subunit Monomeric GTPases: large family of small GTP binding proteins of which Ras is a member -proteins that bind GTP Ras becomes activated by: GEF: Guanine nucleotide exchange factor GAP: GTPase activating proteins they help activate the monomeric GTPases RTK and Ras signaling Ras can activate the MAPK signaling pathway (last kinase in chain) -often see a change in gene expression but can be change in protein activity MAP kinase kinase kinase phosphorylates MAP kinase kinase which phosphorylates MAP kinase 30% of cancers contain mutations in Ras that inactivate the GTPase activity What happens to Ras if there is no GTPase activity? (check this) -Cellular survival and/or proliferation common response Ras can activate PI-3-kinase-Akt signaling PI-3-kinase-Akt can help suppress apoptosis (check? she fucking babbled for like 5 minutes) AKT promotes cell survival -Bad indirectly stimulates apoptosis by binding and inactivating Bcl2 -Bcl2 suppresses apoptosis Cellular Crosstalk When the target protein is fully phosphorylated, it triggers a cellular response fig 16.43 What would happen if only signal A were present? no response What would happen if A and D were both present? yes response What would happen if B and C were both present? no response ~switching powerpoints to Cell Signaling - Disrupting the Signal~ Going to talk about the Huntington’s disease paper and go through how they figured this out Huntington’s Disease (HD) Onset 35-45 years Neurodegenerative disease Muscle coordination affected Cognitive decline-> dementia Lethal: life expectancy 55-65 yrs Autosomal dominant disease 1:10,000 people in US No cure; treatments just help relieve symptoms Huntingtin Gene (HTT) Trinucleotide repeat (CAG) is expanded in the coding region of HTT gene resulting in expanded string of glutamines (Q) if you are Healthy: 10-26 repeats if you have HD: >37 Longer the expansion; earlier onset of HD symptoms mutHTT adopts pathogenic conformation resistant to degradation; accumulates in cell Huntingtin (HTT) s i s i h t e s u a c e b t n a t r o p m i s i h t i w t c a r e t n i t ’ n d l u o w t i d n a e b u t e h t n i , d a e b e h t d n a P F G h c i h w T T H o t d e h c a t t a s i P F G -If you are not allowing PPAR to work the way its supposed to you are going to inhibit PPAR transcription Formal Hypothesis: Mutant HTT (poly-Q-expanded) binds PPARδ and prevents it from activating gene transcription. So another test to test this hypothesis ^^^ Knock-in mice expressing full length mouse HTT protein with different polyQ lengths ST-HdhQ7/Q7 ST-HdhQ111/Q111 Expanded glutamines (HD) They compare expression of genes in both -the genes that they know are regulated by PPAR -Which mouse should have higher levels of PPARδ dependent gene expression? -the Hdh Q7/Q7 because this is the WT mouse and PPAR in this mouse is carrying out its normal function -in Hdh Q111/Q111 they have Htt so PPAR can’t function normally and they have LOWER levels of expression Hypothesis: Mutant HTT (poly-Q-expanded) is interacting with PPARδ and preventing it from activating gene transcription. Loss of PPARδ gene expression is contributing to mitochondrial abnormalities, neurodegeneration, and motor dysfunction If this is true, what would you expect to see in mice that lost PPARδ activity through other mechanisms? -you should get the same phenotypes as Htt (mitochondrial abnormalities, neurodegeneration, and motor dysfunction) Testing this: Knock-in mice expressing dominant negative PPARδ -Dominant negative PPAR (DN PPAR) is another thing that prevents PPAR from functioning properly PPAR + RXR —> activate gene expression PPAR + DN PPAR does not bind to RxR —> no gene expression looking at weight r e i v a e h s i T W t n a n i m o d e h t - r e l l a m s s i : N D - motor function (ledge test) -dominant negative: lost balance more Closing score -Dominant negative: clasped their hind legs more Kyphosis (hunch back) -dominant negative: more prone to falling -dominant negative: happens more quickly (respective to time) Hypothesis: Loss of PPARδ gene expression is contributing to mitochondrial abnormalities, neurodegeneration, and motor dysfunction ! s i s e h t o p y h e h t s t r o p p u s Measured size of mitochondria (mitochondrial abnormalities) dominant neg: have smaller mitochondria and less ATP production Counted neuron numbers (neurodegeneation) dominant neg: decreased numbers of nuerons Conclusion: Interfering with PPARδ function in mice can phenocopy HD. Therefore PPARδ dysregulation is a key in HD pathogenesis “We theorized that if impaired PPAR-δ function is contributing to HD pathogenesis, then an attractive treatment option would be to agonize PPAR-δ.” y d u t s m o r f - -agonize PPAR - means to stimulate PPAR -maybe if you add in a ligand for PPAR delta then you can stimulate PPAR transcription genes and help hunting tons disease “Of the various possible PPAR-δ agonists, we opted to use KD3010, as this PPAR-δ agonist is potent and specific, crosses the blood brain barrier and was approved for use in humans in a Phase1b metabolic disease safety trial in which no incidences of side effects were reported.” -This is important because you can’t have the agonist making side effects occur KD3010 PPAR-δ Agonist HD = has huntingtons disease Non-Tg control = non HD mouse -just a normal mouse like WT What is the vehicle control? -whatever your drug is dissolved in ) i l l i v o r c i m a i l i c ( ) e p a h s l a c i r t e m m y s s a ( l l e c e h s t i n u b u s t ) d n e r e h t o a t e b d n a d n e a h p l a s t n e m a l i f f o y t u b d n P T G o t d m r o f T n i e r a s t i n u b u s e l b u l d n u o b P T G / P T A : m r o f d n u o b P D G / P D A : m r o f ) e u l b ( s e l u b u t o r c i m e r o h c o t e n ) d e r ( s e l u t u b o r c i m r a l o p r e t t r o p s n a r t l a n o i t c e r i r e m i d l i o c d e l i o c a d n a h t a e d l l e c d e s a e r c n i , n o i Nucleation Elongation Stabilization Disassembly Cross-linking