Cell bio study guide EXAM 3
Cell bio study guide EXAM 3 BIOL 30603
Popular in Molecular, Cellular, and Developmental Biology
Popular in Biology
verified elite notetaker
This 28 page Study Guide was uploaded by Mallory Notetaker on Saturday April 2, 2016. The Study Guide 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 131 views. For similar materials see Molecular, Cellular, and Developmental Biology in Biology at Texas Christian University.
Reviews for Cell bio study guide EXAM 3
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 04/02/16
Cell Bio Study Guide Exam 3 Glycobiology Glycobiology: Field of biology that studies the structure and function of glycoconjugates and the proteins that specifically interact with them Monosaccharide cannot be hydrolyzed into a simpler unit (CH2O) -found in a ring structure Sugar derivatives are formed when the hydroxyl groups of simple monosaccharides are replaced by other groups -like an amine group -glucosamine, glucuronic acid Disaccharide is 2 monosaccharides joined together oligosaccharides: short chains of sugars (2-10) complex oligosaccharide: non-repetitive sugar sequence -branching can occur in oligosaccharides or polysaccharides -branching occurs because monosaccharides have several free hydroxyl groups that can form links Polysaccharides are negatively charged so they attract water -gel-like consistency (main component of mucus) Glycoconjugate: covalent addition of one or more sugars to a protein or lipid -glycoprotein or glycan (basically means its on the outside of the cell) -1/2 of eukaryotic proteins are glycosylated -glycoconjugates are anything that has a sugar attached to it Glycoprotein vs Proteoglycan both have sugars attached to a protein Glycoprotein is much smaller -so… its an oligosaccharide linked to protein -little bit of sugar on your protein Proteoglycan is much bigger -contains polysaccharide chains called GAG chains -little bit of protein on your sugar GAG chain = glycosaminoglycan chains -long, unbranched sugar chains composed of repeating disaccharide units one of them HAS to be an amino sugar -often sulfated; highly negatively charged GAGS form gel-like matrix because of their negative charge -GAGs occupy a huge volume relative to their mass. -form porous hydrated gels that helps the body withstand compressive forces -found in joints (extracellular matrix) Proteoglycans = GAG chain + protein -if the GAG chains is attached to a protein it was assembled in the golgi You can have a GAG chain that isn’t attached to a protein -example: hyaluronan is a gag chain that is assembled in the plasma membrane -its found in beauty products because when you put it under your skin, the negative charge attracts water and this is filler and no wrinkles Focus is now on GLYCOPROTEINS Functions -protect a protein from degradation -hold it in the ER until protein is properly folded -transport signal for protein trafficking -form glycocalyx sugar coating involved in cell to cell recognition and protection Protein Glycosylation in ER -sugars are covalently added to proteins in ER -pre-formed precursor oligosaccharide is transferred to proteins in ER -N-linked because the sugar hooks onto the NH2 side chain on asparagine Trigger sequence is either: Asn-X-Ser or Asn-X-Thr Formation of the precursor oligosaccharide (occuring in lipid bilayer of ER membrane) 1. it is assembled sugar by sugar onto the carrier lipid dolichol 2. the first sugar is put on and uses a high energy pyrophostate bond 3. then 2GlcNac and 5 Man are added which causes it to flip into the membrane 4. 4 more mannose and 3 more glucose are added inside the membrane Final pdt: 3 glucose 9 mannose 2 GlcNAc Oligosaccharyl transferase is hanging out near where the protein is being brought in the ER -it is scanning the sequence of protein looking for Asn -it will attach the oligosaccharide onto protein using pyrophosphate bond energy -location of glycoslyation doesn’t really matter How glycosylation helps with protein folding: last two glucose will be trimmed off of the oligosaccharide -the remaining glucose will attach to calnexin and allow the protein to be held in the ER membrane while it gets properly folded -glucosidase: remove a last glucose and let properly folded protein leave ER -if not properly folded glucosyl transferase will add a glucose to it again and it will go bind to a chaperone protein again going to golgi after ER no diversity in protein glycosylation in the ER -only in golgi gets really complicated because not all proteins are going to interact with the same sugar groups Two different routes -high mannose oligsacharide: its trimmed a lil in the golgi but no sugars are added there -complex oligosaccharide: trimmed and sugars ARE added in golgi Process of putting together complex oligosaccharides -all 3 glucose came off in ER along with 1 mannose -in golgi now some mannose are trimmed (3) -if it leaves now its a high mannose oligo Exposure to enzyme Endo H (way to see if it is high mannose or complex oligosaccharide) -endo H can degrade the sugar if its just the mannose but if it has the additional sugar on it, it cannot -high mannose oligo = Endo H sensitive -complex oligosaccharide = Endo H resistant Glycosylation that occurs in golgi is called O-linked -when the sugar is added to the Oxygen on threonine or serine - not just one sequence like in N linked proteoglycans are always o linked glycosylation is so important in making sure proteins leaving the golgi go where they are supposed to go example: mannose 6-phosphate tag is added to lysosomal hydrolyases in golgi (M6P tag directs them to lysosome) mannose got added in the ER (mannose 6-phosphate tag) the picture is showing that a protein with a specific sugar tag came to golgi and interacted with the specific protein (GlcNAc phosphotransferase) and a GlcNAc with a phosphate tag is added and then theres an additional enzyme that comes and removes the GlcNAc -GlcNAc just used to add phosphate Transport of lysosomal Hyrolases when in the lysosome, the mannose 6 phosphate dissociates because it was only used to get the protein there (lysosome hydrolase) I-cell disease Consequence: tags can’t be added to mannose lysosome doesn’t work because it has no lysosomal hydrolases and you get protein aggregates in the cell -so the proteins get secreted outside the cell and into the blood (because they don’t know where to go , no tags) -fatal by 6 years old Bird Flu (influenca and sugar linkages) Why it achieve human to human transmission the receptor that the virus recognizes is glycosylated -for the flu receptor the last two sugar in the complex oligosaccharide are -SIALIC ACID -GALACTOSE -it matters about the linkage between these two… the sialic acid can link in a 2,6 linkage or a 2,3 linkage human flu recognizes 2,6 linkage Bird flu: recognizes 2,3 linkage In birds all of their lung cells express sugar chains have a 2,3 linkage between the sialic acid and galactose so they have evolved to recognize that in humans the upper respirator tract cells expressed 2,6 linkages and only in the very lower parts were 2,3 linkages detected Glycobiology II Sialic acids and Glycans Glycoproteins are a lot smaller than proteoglycans Question: protein is glycosylated, it doesn’t have to have begun in the ER, can happen only in golgi -if it is O linked, it starts in the golgi -if you had serine, it has to have happened in the golgi -sequence in the ER is Asn-X-Ser, Asn-X-Thr Sialic acids are the most common terminal sugar in mammalian cells (not found in plants or anything else) -sequences can vary but ALWAYS last is sialic acid -Negatively charged NOT found in plants, invertebrates or prokaryotes EXCEPT for some human pathogens -human pathogens hide themselves by coating themselves in siliac acids -if you delete genes coding for these you get embryonic development issues Kinds of sialic acids: Neu5Ac found in humans Neu5Gc found in all other mammals The reason humans have different than mammals is because Neu5Ac is the precursor to Neu5Gc -CMAH is the gene that converts Neu5Ac to Neu5Gc -humans lost this gene so we don’t make Nue5Gc Which type would you expect to see in?… -Beef you expect to see both types because yes its a mammal so Gc but ALSO Ac is the precursor to Gc so it can have both -carrots have neither Humans lost the CMAH gene as a way to avoid infection by Plasmodium species that cause Malaria Malaria: caused by plasmodium sp (parasites) and it invades the red blood cells -synchronized lysing of RBCs is what causes host to have alternating chills/fever -spread by anopheles mosquito Plasmodium binding to RBC critical point in lifecycle -if this doesn’t happen, nothing will happen (won’t get disease) 1. Plasmodium enters through mosquito bite 2. Plasmodium travels to liver and then to bloodstream where it infects RBCs 3. Plasmodium gametocytes ingested by mosquito 4. Lifecycle completed in mosquito no human to human transmission There is plasmodium that will only infect humans and some that will only infect apes -Plasmodium falciparum — infects humans -Plasmodium reichenowi — infects great apes NO CROSSING OVER OF THESE -even though Plasmodium species seem nearly identical BUT Aotus monkey is susceptible to Plasmodium falciparum like humans -Why? -because Aotus monkey’s Red blood cells have peak at Neu5Ac peak (fluorescence) Malaria and RBC binding Plasmodium protein EBA-175 — binds to glycoprotein on RBCs Pf-EBA175 from P. falciparum (infects humans) Pr-EBA175 from P. reichenowi (great apes) Hypothesis for why we don’t have crossing over of the infectious disease malaria: Pf- EBA175 will preferentially bind Neu5Ac coated RBCs and Pr-EBA175 to Neu5Gc RBCs -this supports the hypothesis, there is preference Plasmodium event — when plasmodium split into the Pf and Pr (fig 1) Our biochemical pathways cannot tell the difference between Neu5Ac and Neu5Gc -so if you eat food that has Neu5Gc your body will use that -this means our cells have Neu5Gc incorporated in their glycans and therefore on their surface But our immune system can tell the difference -it makes anti-Neu5Gc antibodies Neu5GC sugars on human endothelial cells are labeled with red fluorescent antibodies -when are these being made -looked at babies and mothers -babies were being breast fed so they didn’t have any Neu5Gc antibodies -when they started eating real solid food, they had them on their RBCs antibody: protein produced by B-cells that binds “foreign” molecules — actually bind to antigen because antigens are usually foreign Antigen: molecule that induces immune response (usually foreign) once you have an antibody bound to an antigen on bacterium it is a big kill me sign -the antibodies do not kill microbes themselves, if an antigen binds to antibody it will phagocytose and send signals to immune system Response to tissue injury/infection -Recruitment of WBCs and serum proteins (antibodies, complement) -Microbial destruction through phagocytosis, complement mediated killing and release of antimicrobial chemicals (peroxide) Acute inflammation: initial inflammatory response Chronic inflammation: prolonged inflammation -there can be damage to host cell -when macrophages are being recruited to the same space and they shouldn’t be Chronic inflammation has been linked to cancer and atherosclerosis which is the build up of plaque in the arteries Why don’t our cells make Neu5Gc? -loss of CMAH Where is Neu5Gc coming from? -food Why do our cells incorporate Neu5Gc? -because they can, they don’t tell the difference Why do we make antibodies against it? -because they can tell the difference -what are the consequences of this? making it against your own cells The worry is that making anti bodies against self is causing chronic inflammation (linked to build up of plaque) Why do we make antibodies against it? -what are the consequences of this? making it against your own cells Hypothesis: Endothelial cells with Neu5Gc on their surface would be more likely to attract phagocytic cells and complement due to antibodies against Neu5Gc. -THIS TEST IS SHOWING HOW THIS IS RELATED TO CAUSING HEART DISEASE OR ATHEROSCLEROSIS Growing cells in dishes -fed cells different sugars (Ac or Gc) -then added Neu5Gc antibodies — which should recognize only the Gc sugars -if their hypothesis is correct you should get a higher recruitment of phagocytic cells with Gc sugars Human serum= 20% antibodies -how you get antibodies They took the blood of people and their levels of Gc antibodies varied a lot -this can be caused by what they eat, meat or not -they took somebody with high levels and low levels -they took their endothelial cells that were fed Gc or Ac -and they looked at phagocytic recruitment in their specific cells If the hypothesis were correct and you had Ac-fed exposed to +S30 you still have low levels of phagocytic cells -ITS NOT ENOUGH TO JUST HAVE THE GC SUGARS INCORPORATED IN YOUR CELLS -you need the high level of Gc antibodies to have the high levels of recruitment of phagocytic cells Does this prove that the presence of Neu5GC on endothelial cells contributes to atherosclerotic plaques? -no (because its not just the presence) -previous research has shown an association between phagocytic cells and plaque Now this test is about how sialic acids can cause or not cause cancer you have a Wild Type mouse and a -/- mouse — meaning you have a knock out of both CMAH genes WT mouse will make Nue5Gc because its a mammal and has CMAH gene Which mouse will make αNeu5Gc antibodies if exposed to Neu5Gc? A) WT B) CMAH -/- C) Both D) Neither NOT BOTH because WT normally makes Neu5GC so it won’t make antibodies against your own stuff but if you inject Neu5Gc into a CMAH -/- they will -because -/- didn’t have Gc before and now they are exposed to it so it is considered foreign and will make antibodies against it -AGAIN, WT mouse will NOT make antibodies because its normal self is Nue5Gc Whole Idea of study: Antibodies —> chronic inflammation —> cancer If this is true, which mouse, WT or Cmah -/-, would be more likely to develop inflammation and eventually cancer after exposure to Neu5GC? -the CMAH -/- To test it: they put tumor cells expressing Nue5Gc in mice and looked at the size of the tumor growth -the bigger tumor was found in -/- mouse due to that mouse making anti bodies causing inflammation and cancer Exposure to Neu5Gc in animals that lack CMAH leads to production of αNeu5Gc antibodies Hypothesis: In animals lacking the CMAH gene, exposure to Neu5Gc rich foods in the presence of αNeu5Gc antibodies will lead to increased inflammation and development of spontaneous cancer. Data said that the mouse that was injected with Neu5Gc antibodies and was fed Neu5Gc rich foods, had way higher levels of inflammation. Babies levels of Nue5Gc are none until they start eating real food (not breastfeeding) so if we don’t eat Nue5Gc food we won’t make antibodies against them and we would reduce our risk for cancer. Metabolism and Mitochondria You release the same amount of energy when you directly burn it or a stepwise oxidation -cells do a stepwise release so that it is able to capture the energy along the way 3 stages of how the cell metabolizes food first stage- breaking down foods to different subunits, amino acids, glycerol second stage- glucose goes through glycolysis and makes pyruvate (turned into acetal CoA) third stage- break down of acetal CoA and oxidative phosphorylation Oxygen is not required for glycolysis -you only need that during oxidative phosphorylation (citric acid cycle) 2 mechanisms for ATP generation -1. Substrate-level phosphorylation - when a phosphate from a molecule is added to ADP happens during glycolysis -2.) oxidative phosphorylation - through electron transport chain Glycolysis 1 molecule of glucose -> 2 molecules of pyruvate 2 ATP consumed 4 ATP produced 2 NADH produced Net gain: 2 ATP and 2 NADH and 2 molecules of pyruvate Substrate level phosphorylation (occurs at steps 6 and 7 of glycolysis) -(6) first NADH is made -(6)then a high energy phosphate bond is made and when it is broken… -(7)then the cell can transfer the phosphate to ADP to make ATP This addition to ATP (from the precursor of pyruvate) is energetically favorable You should know information presented in the last 3 slides on glycolysis, but you do not need to know detailed steps in panel 13-1 -whats happening to sugars -where ATP is being consumed and produced At the end of glycolysis 2 molecules of pyruvate have been generated -then pyruvate can go into mitochondrial matrix and go through citric acid cycle (but have to have oxygen) -if none is present then it undergoes fermentation Lactic acid is produced (in muscles when you are running out of oxygen) from pyruvate Yeast -turning pyruvate into ethanol Is ATP generated during these fermentation steps (i.e. pyruvate -> lactate or pyruvate -> ethanol)? -NO -In glycolysis? YES just not in fermentation step T or F- the reason cells undergoes fermentation instead of just discarding pyruvate as a waste product is because fermentation products such as lactate shown here can later be broken down for energy once oxygen is present? FALSE- that is not the reason fermentation happens -the reason is to regenerate the NAD+ -during glycolysis NADH is made and fermentation regenerates NAD+ -allows glycolysis to continue BUT USUALLY O2 is present -pyruvate is pumped into mitochondria and converted to acetyl CoA Pyruvate -> Acetyl CoA -one of the carbons of pyruvate leaves as CO2 generate: 1 CO2 1 NADH Note: This is not the only way for the cell to generate Acetyl CoA. Can also generate if from fatty acids—see Fig. 13.11 in textbook. Glucose is fully oxidized by the end of the citric acid cycle and released as CO2 2 molecules of CO2 3 NADH molecules 1 FADH2 1 GTP are all generated for every one molecule of glucose you get 2 molecules of pyruvate and then 2 molecules of acetal coA Glycogen is a long polymer of glucose and stores energy or fatty acids can be converted to acyl coA T or F the cell undergoes glycolysis and the citric acid cycle for the sole purpose of producing ATP and NADH and other electron carriers that will eventually be used during oxidative phosphorylation? -FALSE -Ex: pyruvate is the substrate for several enzymes -nucleotides are derived from glucose 6 phosphates Final stage: Oxidative Phosphorylation -requires a membrane -in eukaryotic cells: inner mitochondrial membrane -in prokaryotes: just their membrane Chemiosmotic coupling Where are these electrons originally derived from? -from glucose (being oxidized) Energy of electron transport is used to pump protons across membrane -this creates a gradient and the H’s come back in and go through ATP synthase THEN energy in the proton gradient is harnessed by ATP synthase to make ATP Mitochondria -Location of oxidative phosphorylation electron transfer + ATP formation -Evolved from bacteria engulfed by ancestral cells Allows for MUCH more efficient use of glucose -Glycolysis + fermentation -> 2 ATP -Glycolysis + oxidative phosphorylation -> 30 ATP Without mitochondria, unlikely that complex multicellular organisms could have evolved! The inner membrane is just like a cell membrane is selectively permeable Oxidative Phosphorylation -Consumption of O2 -final thing that accepts those electrons is oxygen (turns into H2O) -Addition of phosphate group to ADP to form ATP Electron Transport System (ETS) -NADH dehydrogenase complex: accepts electrons from NADH and pumps out H+ -Cytochrome C reductase complex: uses energy received to pump out more H+ -Cytochrome C oxidase complex: pumps out H+ but also produces water by transferring electrons to oxygen ETS is really important because it powers ATP synthase -100 ATP being made per second 2 gradients being made -charge gradient (voltage gradient) -pH gradient (lower concentration of H to Higher) Both will contribute to the proton motive force -the stronger this is, the more ATP can be generated This is also used for coupled transport to bring things in the mitochondrial matrix pyruvate and phosphate molecules are negatively charged so naturally they don’t want to come in the mitochondrial matrix (which is already negatively charged) -as hydrogen ions are coming in the matrix down their gradient is used to bring these in this isn’t very common in mitochondria but it is in prokaryotes Reduction is gaining an electron oxidizing is losing an electron look at which species lost or gained an H Each electron transfer in ETS is a redox reaction NAD+ is oxidized NAD+ will have a low affinity for electrons because it wants to give up electrons so it will have a lower affinity than the complex its giving it to oxygen has highest affinity of them all we don’t go from NADH to oxygen because its too big of a voltage difference (1140) that it would almost be explosive, cell can’t handle it cytochrome c oxidase complex holds the oxygen that the electrons will go to very tightly because the oxygen needs 4 electrons to attach to 2 hydrogens for it to form water and if it gets released before this, you get super oxide forming and thats bad, causes damage to mitochondria Cytochrome C oxidase removes electrons from cytochrome C and transfers to O2 (4e- +4H+ + O2-> 2 H2O Complex must bind very tightly to O2 until it has received all 4 electrons otherwise superoxide can form—highly reactive -important that oxygen is held tightly until process is finished Formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2) leads to oxidative damage of mitochondrial proteins, membranes, DNA -if you have high levels of ROS being produced in mitochondria there is lots of damage = BAD -this may be associated with cell aging -sometimes cytochrome c can get released from the cell which can trigger apoptosis -hydrogen peroxide can be formed from superoxide prokaryotes can undergo anaerobic respiration -diffference: O2 not only molecule (prokaryotes use some other molecule) that can serve as terminal electron acceptor in ETS. -However O2 most efficient—more ATP can potentially generated BUT Benefits of anaerobic respiration- Able to grow in environments w/out O2 No reactive oxygen species are generated Prokaryotes that use anaerobic respiration generally very slow growing but have unique evolutionary niche. Cell Signaling hydrophobic signals use intercellular receptors Extracellular signals can do two things: -alter protein function —> alter cytoplasmic machinery —> alter cell behavior -alter protein synthesis —> alter cytoplasmic machinery —> alter cell behavior Which will be faster—altered protein function or altered protein synthesis? protein function - almost immediately, phosphorylation example Intracellular receptors -when extracellular signal molecules are small enough or hydrophobic enough to cross plasma membrane -Ex: Steroid hormones Ex: Dissolved gasses Intracellular Receptors: Nuclear Receptors -they bind steroid hormones -Huge impact on physiology and development Pass through the membrane and meet receptor in the cytosol, if its unbound it is inactive -binds and is active and then moves to nucleus and activates gene expression (binds to specific promoter sequences to activate transcription) nuclear receptors usually make transcription happen Intracellular Receptors: intracellular enzymes Some dissolved gases can diffuse across the membrane and directly regulate the activity of intracellular proteins example: NOS makes a gas (NO) which goes and binds to the enzyme guanylyl cyclase which makes cyclic GMP which then goes and stimulates other proteins within the cell, in the case of muscle cells it can cause cell relaxation FROM PICTURE Which are the extracellular signaling molecules in this figure? -nitric oxide and acetylcholine Which are the intracellular signaling molecules in this figure? -cyclic GMP (made inside the cell and affects other things inside the cell) Which are the receptors in this figure? -acetocholine and guanylyl cyclase example: Viagra blocks the enzyme that degrades cyclic GMP. Why is Viagra effective in treating erectile dysfunction? -no relaxation of the smooth muscle cell so that creates vasodilation and more blood flow to penis Extracellular Receptors Majority of signaling molecules are extracellular receptors -after it binds on the outside -Intracellular signaling molecules pass the signal -Effector proteins carry out the action to produce a cellular response -another name for this is a signal transduction pathway Signal transduction is just when its passing signals to the next Intracellular Signaling Pathways (after the signal is received by extracellular receptor) May have multiple functions 1.Relay a signal 2.Amplify a signal 3.Integrate multiple signals from more than one signaling pathway (can have excitatory signals and inhibitory signals) 4.Distribute the signal to more than one effector protein Molecular Switches Intracellular signaling proteins have to be able to turn on/off in order to control signaling 2 major classes signaling by protein phosphorylation signaling by GTP binding proteins Two cells that express the same receptor will respond in the same manner when the receptor is stimulated? -FALSE -due to different signaling pathways Eukaryotic cell signaling IS REALLY complex 3 major classes of cell surface receptors -ion-channel-coupled receptors -g-protein-coupled receptors -enzyme-coupled receptors The number of different types of receptors is greater than the number of different extracelullar signal molecules -true, there is more receptors than there is signaling molecules -ex: acetylcholine activates different receptors in heart pacemaker cell and salivary gland cell g-protein coupled receptors are used for smell -mice have more of these G-protein-coupled receptors -Huge receptor class—more than 700 GPCRs in humans alone -All have similar structure—7 pass transmembrane receptor -All are coupled to G-proteins -receptor interacts with g protein . G-proteins 3 subunits-α, β, γ tethered to membrane α subunit bound to GDP when unstimulated -then when a ligand binds it allows for a conformational change that allows for the exchange of GDP with GTP Process of G-coupled protein receiving a signal -Signal molecule binds GPCR -GPCR undergoes conformational change -G-protein alters conformation -Alteration of α−subunit changes affinity for GDP and GDP dissociates -GTP exchanged for GDP -Conformation changes of g protein -α and βγ subunits now active (you often times have the disassociation of the Beta and Gamma parts) (can go on to interact with target protein) - Alpha subunit Interacts with target proteins -This can activate or inactivate the target protein—depends on G protein Gs—stimulates target protein Gi—inhibits target protein -GTP hydrolyzed to GDP -Subunits dissociate from target protein -α,β,γ subunits reassociate forming inactive G protein that is ready to couple once again with GPCR she said the g protein is shut off by intrinsic ability of g protein G protein Targets: ion channels example -Ach released by nerve cells -Ach binds GPCR on heart pacemaker cells -Activated βγ complex binds K+ channel -K+ channel opens -Increases membrane permeability to K+ -Makes it more difficult to electrically activate cells -Heartbeat slows 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 When Akt is activated it goes and causes phosphorylation of Bad AKT promotes cell survival -Bad indirectly stimulates apoptosis by binding and inactivating Bcl2 -Bcl2 suppresses apoptosis Bad and inactive Bcl2 are bound together -then get survival signal from active Akt when you phosphorylate Bad is lets go of Bcl2 and Bcl2 is now active and can go inhibit 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) Medium-sized spiny neurons primarily affected Aggregates of mutHTT forms inclusion bodies (orange) in nucleus Molecular Impacts 1. Transcriptional dysregulation 2. Impaired protein degradation 3. Altered protein folding 4. Disrupted neuronal circuitry 5. Mitochondrial dysfunction Grew GFP-HTT-25Q in petri dish -then you put gene on a plasmid, then you clone these(GTP-HTT) into the plasmid and you put plasmid into the cell by transfection -collect the cel lysates (get ride of the cell membranes and collect proteins in the cell) How would scientists separate out only the GFP-HTT-25Q proteins? -then you can do immunopreciptitation (IP) using an GFP specific antibody (alpha GFP) -at the bottom of the anti body it will have a little bead -it will pull down GFP-HTT-25Q and whatever it is bound to (in centerfuge) -you would pull down any proteins its interacting with If HTT is in the nucleus, what is the green protein likely to be? -a transcription factor, makes sense because they have done staining to know that Htt is in the nucleus so it would be interacting with a protein that is in the nucleus which most are transcription factors -and at some point they are going to bind to DNA What type of protein? What do all transcription factors bind? -DNA -this is not random, very specific sequences this is important because this is how they could tell which transcription factor it was Transcription factors bind specific DNA response elements. These response elements are known for most transcription factors -they had an array of lots of DNA sequences, all containing known response sequences -where is this green protein binding? they found that it is binding to a response element recognized by PPAR-delta Peroxisome Proliferator Activated Receptor (PPAR) 3 types PPAR-α PPAR-δ PPAR-γ these ligands can be fatty acids, etc. then PPAR protein goes into the nucleus and will heterodimerize with RXR (retinoid X receptor) and then activate transcription after it recognizes its specific sequence -PPAR is a nuclear receptor, its not a steroid receptor but acts similar to them PPAR + RXR Bind DNA response elements w/ sequence AGGTCANAGGTCA (don’t have to memorize_ after that it can activate transcription They think that HTT interacts with PPAR delta to prove this: -they put GFP in the petri dish -also put Flag-PPAR-delta -they collect the cell lysates -they do IP GFP What would you expect to see? -just GFP (because thats the one with the bead and it wouldn’t interact with PPAR or anything else) Because … you would not see B (GFP and PPAR interacting) because -GFP interacts with Htt first THEN PPAR will interact with GFP -in this example no Htt was added so no interaction Now what? How do you actually “see” which proteins are in the tube? What technique can you use? -Now since they added Htt and that attaches to GFP and the bead, in the tube you will see GFP AND PPAR because the Htt is interacting with the PPAR and it is pulled to the bottom of the tube -NOW you can use western blot technique Western Blotting - for protein separation (by size) After running total cellular protein and proteins after IP on a gel -you will see all proteins plus target proteins so to see the target proteins… -you add primary antibodies against PPAR delta (immunoblot for PPAR) -you add a second antibody with a tag that lights up (detect that signal) -then you will get bands that correspond with your target protein Protein we are interested in detecting is PPAR delta All steps : 1. collect protein lysates 2. IP GFP 3. western Why immunoblot with Flag antibody -flag with ultimately recognize the PPAR delta because it recognizes flag and flag is attached to PPAR (Flag-PPAR-delta) In this actually setup they ran three conditions 1. GFP + Flag-PPAR-delta 2. GFP-HTT 25 (glutamine repeats or Q) + Flag-PPAR-delta 3. GFP-HTT 104 Q + Flag-PPAR-delta KEY: input is going to be everything (all proteins before you do IP) (called input) the right half (IP: GFP) after positions: 1 = GFP + FLAG-PPAR-delta 2 = GFP-HTT 25Q + FLAG-PPAR-delta 104Q 3 = GFP-HTT + FLAG-PPAR-delta On the left half (input), Why are there bands with all 3 GFP proteins? -there are bands by IB: FLAG for each trial condition because it is detecting the presence of FLAG-PPAR-delta which was added in each and had not yet been separated out by IP: GFP bead and brought to the bottom After IP: GFP what positions should have bands? -2,3 showed bands -this is because IP: GFP separates out GFP by bringing it to the bottom (due to the bead) and in position 1 there is no band because the band signifies the presence of PPAR and as the key above shows, position 1 has only GFP with no HTT so it would not interact with PPAR -positions 2 and 3 do have bands because GFP is attached to HTT which allows for the interaction between GFP and PPAR and IB:FLAG is detecting PPAR that is separated out with GFP and is lighting up (showing bands) So now they have proved there is a specific interaction between HTT and PPAR -and this extra long repeating CAG sequence in the HTT gene causes PPAR to not function properly -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 non-expanded glutamines (WT) —- normal ST-HdhQ111/Q111 Expanded glutamines (HD) —- HTT 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 -the dominant WT is heavier -DN: is smaller 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 supports the hypothesis! 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.” -from study -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 -showing that just injecting a saline solution is not having an effect What can they conclude? How effective was KD30101? -not 100% effective but it does bring the neurological dysfunction score down from non treated What did these scientists discover that was novel? The big thing they didn’t know before: huntingtons gene interacts with PPAR delta They showed that if you inhibited PPAR you got a similar phenotypes as huntingtons showed you can help it through adding an agonist ~switching to Cytoskeleton~ Cytoskeleton -cell division (forming spindle apparatus) -stable structures (cilia micro villi) -polarization of the cell (assymmetrical shape) -mechanical strength -Motility and phagocytosis -ability to fight off bacterial infection Cytoskeleton filaments 3 major families -Microtubules: intracellular transport -Actin: Cell shape/locomotion -Intermediate Filaments: mechanical strength Accessory proteins also contribute The cytoskeleton function as highways in the cell but are NOT permanent structures in the cell, more like an ant trail, it is able to move and adapt if something is put in its way it will just go around Cytoskeleton Organization The reason you have this dynamic ability in your cell is because of small soluble subunits that make up cytoskeletal structure. they go back in forth from being individual units and being large filamentous polymers -disadvantage: this takes energy Advantages: rapid diffusion of subunits rapid structural reorganization Tubulinforms Microtubules Tubulin subunit= heterodimer made of α and β subunits (these stack up to make the large filaments mentioned above) Tubulin heterodimers = microtubule subunit -Binds GTP -plus/minus ends (polarity) (due to the alpha end and beta other end) -Microtubule = 13 protofilaments -protofilaments are a straight line of alternating alpha and beta subunits -then 13 of those get side by side to make microtubule -hollow inside Actin - thin and flexible Monomer -but still have plus and minus end because the differing shape of ends Binds ATP instead of GTP Actin filament -> 2 protofilaments wind together, tight bonding Filaments twist around each other in right-handed helix Crosslinking between filaments actin is thin and flexible microtubules are stiff different cells will express different types of tubular and actin ex: alpha actin only found in muscle cells Tubulin binds GTP -alpha subunits hides GTP deep in its center so it never gets hydrolyzed to GDP -beta binds it on the outside so it DOES get hydrolyzed -this is important for overall stability of filaments Actin binds ATP —ATP in the center but can be hydrolyzed When new subunits are getting added on, it will add when it is in the GTP or ATP state Filament Growth -you can add subunits to either + or - end but -PLUS (+) end has faster growth of subunits than minus (-) end (because of polarity) -the plus end has the beta subunits bound to GTP -After the subunit has been added, you are going to have hydrolysis of GTP to GDP soluble subunits are in T form T form: ATP/GTP bound D form: ADP/GDP bound -polymers are a mixture of T form and D form PLUS side there will be a ATP/GTP cap (which favors growth) -because the ATP/GTP bound subunits are getting added so fast that the hydrolysis isn’t keeping up MINUS side hydrolysis will happen before a T form subunit can be added When it goes from GTP to GDP, you don’t get as strong of a bond, you start to see bending of the subunits -this is why the GTP cap is good for growth -if they are curving away from each other, called shrinking or a “catastrophe” Dynamic instability -When you have rapid growth, the addition starts to lag, the hydrolysis will catch up and then you have rapid shrinking filaments continuously grow and shrink Catastrophe: Loss of GTP cap and rapid shrinking In actin its called Treadmilling -can have an equilibrium between addition and loss so the overall length stays the same Dynamic instability = microtubules Tread milling = actin —Both allow for spatial and temporal flexibility in filament formation Spindle Formation 3 classes of dynamic microtubules: Kinetochore microtubules (blue) Interpolar microbutules (red) Astral microtubules (green) Microtubules contribute to the polarization of the cell because they are polarized so charged things will move one way on one microtubule and the other way on another -directional transport Example: Neuron -All microtubules in the axon point in the same direction—plus ends toward axon terminal Intermediate Filaments (IF) -you have a alpha helical region of monomer (NH2 at one end and COOH on the other) -that monomer interacts with another to form a coiled coil dimer -then two coiled coils get in a staggered tetramer then lateral bonding eventually to be like a rope -because of this there is no polarity -tetramer is the same thing as tublin subunits or actin subunits It is like a rope and very hard to break because of the strong lateral bonds -found in cell subject to mechanical stress example: epithelial cells Keratin is an intermediate filament found in epithelial cells -you can have mutations in these Example of mutation: Epidermolysis bullosa simplex intermediate filaments aren’t making the right connection -any stress on the skin, get huge blisters -can lead to high risk of infection Nuclear lamins - found in nuclear envelope -Nuclear lamina (blue) formed of IFs called lamins -Strengthen inside surface of the inner nuclear membrane -Provide attachment points for chromosomes -Phosphorylation of lamins causes disassembly during mitosis Progeria - advanced aging (hair loss, joint problem) -caused by defect in lamin processing -Nuclear lamina does not form properly -Nuclear envelope does not have enough structural support—abnormal shape -Nuclear instability -> impaired cell division, increased cell death and diminished tissue repair Bacterial Homologues -Originally thought bacteria did not have a cytoskeleton -but FtsZ is tubular like -and there is also actin homologues that determine cell shape in bacteria Accessory Proteins - help regulate the function of cytoskeletal parts -can regulate length, stability, geometry and therefore function of cytoskeletal filaments through accessory proteins roles: Nucleation Elongation Stabilization Disassembly Cross-linking
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'