Lecture #9 - Protein Turnover Flashcards

Question

What mediates intraluminal vescile

Answer 1

Monounbiquitin of cargo mediates its sorting into intraluminal vesciles - Monoubiquination signlas that the protein should be included in inraluminal vescile

Answer 2

ESCRT proteins unlike coat proteins don’t surround the intraluminal vescues BUT instead promote inward formation by pushing into the vescile to cause membrane deformantion and scission ESCRT complex sits at the surface of the endosme and forms spiral like structures that pushes down the surface of the vesicle into the inteirior of the endosome - Once push down the componets that have been sitting on the surface can be recyled and reused

Answer 3

Coat porteins (Cops) = promote budidng into cytosol ESCRT promote budding away from cytosol - Used in Intraluminal vescile formation and retrovial budding ECSRT proteins are recycled (similar to coat proteins)

Answer 4

Retroviruses (HIV) recruit and use ESCRTs to bud out of the cell Hrs/Vps27 sits on the surface of the endosome and recruits the ESCRT machinery to the late endosome HIV Gag protein sits on the plama mebrane mimics Hrs --> recruits the ESCRT machinery to the plasma mebrane along with viral particles, --> ESCRT facilitates budidng of viral particle away from the cytosol for viral budding out of the cell

Answer 5

Autophagy = process that allows cells to break down obsolete parts of itself for disposal or re-use Autophagy occurs during stravation During starvation autophagasomes deliver bulk amounts of cytoplasmic molecules and or whole organelles to the lysomes

Answer 6

Image = autophagasome in EM (see the double membrane srutcuere)

Answer 7

1. Starvation signal that intitaes the phagafore formation (double membrane stcuture) 2. Phagafore becomes the autophagasome - Formation of a double-membrane around cytosolic components, to form an autophagosome - Autophagy invloves enclosure of cytosolic proteins or an orgenalle by a double membrane phagaphore 3. Autophagosome fuses with the lysosome allowing for degradation 4. Lysosomal hydrolases break down the autophagic body --> allowing recycling of molecular components

Answer 8

Used Yeast genetic screen, based on loss of viability upon starvation --> yielded 35 ATG (aka APG) genes Yoshinori Ohsumi Found the compenents of autophagy Autophagy is currently in the research spotlight --> linked to diseases (cancer + neurodegeneration + pathogen infection +aging) Data can be conflicting --> some evidence suggests that autophagy protects against cancer, other evidence suggests it promotes cancer

Answer 9

mTOR = kinase complex on the lysosmes that senses nutrient levels and is master regulator of autophagy - During starvation mTOR interpets signals and intiates autophagy When mTOR is on autophagy is off ; when mTOR is turned off autophagy is turned on - Rapamycin = inhibitor of mTOR and induces autophagy

Answer 10

Autophagy is invloved in almsot eveyrthing Many diseases and physiological states involove autophagy as the major player Image - shows many things that autophagy is involoved in

Answer 11

In addition to starvation induced BULK autophagy (A in image) autophagy can be selective Selective autophagy = removes specifc damaged organelles (ex. peroxisomes/mitocondria)+ protein agregates + pathogens (B in image) - Have mitophagy + Agrgrepagy (protein aagregates) + Xenopagy (bacteria) + pexophagy

Answer 12

Way selective autophagy works – orgenelles or strctures expose some kind of signal that makes them crago for a receptor and they can interact wot the phagaphore forming structures Crago binds to receptors --> recepotor binds to ATG8/LC3 - Specific autophagy RECEPTORs recignize different types of cargo for delivery to the autophagasomes

Answer 13

Autophagasomes and Intraluminal vesicles from the MVB have the same fate --> BOTH end up docking with and fusing woth the lysosome for degredation Image shows Autophagy Vs. Lysosomal degregation - Autophagy = double membrane vs. Enodsomes have 1 memebrane and have intraluminal vesciles

Answer 14

Occurs during stavation Overall - Proteins enter the lysomes by a non-vescular pathway - Proteins use Hsc70 chaparones and a protein transport chanel on the lysosome to enter the lysasome

Answer 15

Uses KFERQ recognition motif - 20% of protein have something that can be recognized as KFERQ 1. KFERQ reocognition motif is reognized and bound to by Hsc70 proteins in the cytosol --> allows for delivery to lysomal membrane protein lamp2A 2/3. The chaperone-protein complex is delivered to lamp2A --> when have crago bound to lamp2A it multimerizes to form a transport channel 4. Protein unfolds and passes through Lamp-2A transport channel inot the lysosome by a Hsc70 chaparone inside of the lysosme 5. A Hsc70 chaperone in the lysosome helps to reel in the KFERQ-protein and the protein is degraded by proteases Hsc70 family direct import of select proteins through a protein channel into the lysosome

Answer 16

CMA may be involoved in many diseases Example - parkinsons --> Mutant alpha-synuclein that accumulates in parkinsones may clog CMA machinery

Answer 17

Protein tag Ubiqtuitin is covaletley attached to lysine residues on a target protein by E1, E2, E3 After a single ubiquitin is added to a protein additional ubiquitin can be added to form a chain Ubiquitin chains >/= 4 are reocgnized by the proteosme and lead to degredation of the target protein with the chain into small peptides

Answer 18

Block Degradation by a proteasome inhibitor drugs If a protein is unstable and then you add a proteosome inhibitor and NOW the protein is stable when the inhibotor is added THEN you know that is is degraded by UPS

Answer 19

If you want to detect ubiquinated forms of protens --> immunoprecipiate the protein and blot with an anti ubiquitin antibdy If proteins has 1 lysine you can see a defined ladder that shows 1 Ubiquitin Vs. 2 Ub Vs 3 Ub being added to the protein - When add the protesome inhibitor you block degradation = now have longer chains of ubiquitn AND the chains get darker

Answer 20

Ubiquitinated proteins are often challenging to detect because: 1. They are transient intermeidates because they will be degraded 2. Have deubiquniating enzumes that chew away at the chains if you don’t inihbit them (hard to inactiave deubiqnating enzymes in vitro)

Answer 21

A clear “Ub ladder” is only sometimes be seen - Often ubiquitinated proteins are heterogeneous in MW Get a gel that represents proteins that have heterogeneous modification of ubiqutin because: 1. Due to varying extents of modification - chain links vary and utilization of lysines vary 2. Varying sites for modification) - have many lysines that are accesible END - See smear on SDS-PAGE - When add protesome inhibiter smear gets darker

Answer 22

E1 = Ubiquitin activating enzymes An E1-ubiquitin conjugate is formed with ATP-dependent reaction --> ATP is hydrlyszed to acivate ubiquitin that is covalentley bonded to E1 --> passes Ubiquitin to E2 ubiquitin conjugated enzymes E2 = Ubiquitin conjugating ezymes --> Transfers the ubiquitin to the traget protein

Answer 23

E3 = Ubiqitin ligase (Specifcity factor + brings substrate together with E2) - Key to target protein recognition E3's recognizes and bind specific target proteins and bind to a subset of E2 = bridge to bring the protein and E2 is close proxiimity to allow for the to tranfer the ubiquitin to the target protein and assists more being able to be added on to form a chain - When repeat process = form ubiquitin chain that leads to degredation

Answer 24

As specificty increases have more genes that are encoded 2 genes code for E1 50 genes code for E2 -> have more genes because E2 is located in different cell compartnemnts and are regulated in different ways >600 genes code for E3 --> May genes for E3 is needed because have so much specificty in the system

Answer 25

1. HECT E3s 2. RING E3s There are not many HECT E3 in cell compared to Ring E3

Answer 26

HECT E3s = undergo transient covalent binding to ubiquitin during transfer of ubiquitin from E2 to substrate/target protein - Forms transient covlent intermdiate with ubiquitin HECT = brindging facts that helps ubiquitin get onto the target protein

Answer 27

RING E3s do NOT form covalent intermediate with ubiquitin when they bring E2 close to the target to allow for transfer to occur - Just brings E2 and the target close enough together for the transfer to occur - Act only as adapters

Answer 28

Proteosome = degrades proteins to peptide fragments - Fragments are 7 amino acids or smaller Two major structural compoennts of 26S: 1. 20S core - Where the proteolytic enzymes are 2. 19S cap - functions in substrate recognition + unfolding + de-ubiquinating + feeding into the 20S core

Answer 29

20S core = stack of 4 7 membered rings with alpha-SU and beta SU - Alpha-SU = structural - Beta SU = inner rings that have the proteolytic active sites facing the inside of the chamber

Answer 30

Beta SU each have distict proteolytic activity (each cleave after acidic or basic or hydrophobic resiludes) - Myriad of activities combine to give the proteosome broad specificity Proteases in the proteasome core are sequestered away from the cytosole - Because the active site is in the camber the protein is degraded as it passes through the chamber

Answer 31

Proteosome inhibitors (MG132) to bind to proteases active site in beta SU inside the proteosome chanel and block activity Proteosome inhibitors are used in cancer - Example - Bortezomib (Velcade) for multiple myeloma - WHY - Because cancer is fast growing cell = need to make proteins --> means they need to degrade those proteins to create a balance between degredation and synthesis (IF inhibit degredatiion then inhibit the growth of cancer cells)

Answer 32

Pore of 20S core is too narrow all allowing a folded protein to pass Proteins are unfolded when they go through the proteosome 20S core and are degraded as they go through the 20S core Image (beigh string) = protein going through the core (being eaten by the beta SU) - Ball at the bottom (below red) = folded protein that needs to be unfoldoed to go through the core and is degraded as goes through the core

Answer 33

Overall - 19S cap regulates substrate entery into 20S core Roles: 1. 19S cap has a Ubiquitic recpetor that recognizes and binds to ubiquinated proteins with chain of >4 ubiquitins 2. 6 AAA ATPases form an unfoldase ring that uses ATP hydrolysis to unfold the protein and feeds the proteins into 20S core 3. Ubiquitin hydrolase ('de-ubiquinase') removes the ubiquin from substate for ubiquitin recycling - Major role of cap = cleave off ubiquitin from teh prptein as it is unfolded/before it enters the core

Answer 34

Ubiquitin = small protein Ubiquitin = covalentley linked to lysine in protein through isopeptide bind between C terminal Gly (Gly-76) carboxylate on ubiquitin and the NH amino group side chain of lysine on target protein Multiple ubiquitins can be linked to the protein in the form of a polyubiquitin chain - For degredation - 1 Ubiquitin Gly carboxylate is linked to lysine on the protein itself AND the Gly carbpxylase (Gly76) on the second ubiquitin forms an isopeptide bind with the lysine48 on the first ubiquitin (repeat this process until form a K48 chain) - K48 chain of >/=4 ubiquitin is recognized by the 19S proteasome cap

Answer 35

1. Ub is “activated” by covalent binding of the E1 ubiquitin activating enzyme 2. Ub is transferred to an E2 ubiquitin conjugating enzyme 3. The E3 ubiquitin ligase which has substrate recognition properties promotes transfer of ubiquitin from the E2 to a substrate lysine residue - E3 binds E2 in close proximity to substated = transfer 1 ubiquitin onto the lys of protein 4. After formation of the first Ub-substrate bond, a second Ub can be conjugated to a specific lysine (typically Lys-48) of the first Ub and leads to the assembly of a polyubiquitin chain on the substrate (forming chain) 5. Substrates bearing polyubiquitin chains of >4 ubiquitin in K48 link are recognized by the 19S proteasome cap, undergoes deubiquitination, unnfolding, and is fed into the 20s core 6. The substrate protein is degraded and Ub is recycled

Answer 36

Ubiquitin can for 7 types of chains Ubiquitin has 7 lysines --> means they can make polyubiquitin chains in different confirmations Examples: 1. K48 chains = >/= 4 ubiquoitin long direct a protein to the proteosome 2. K63 (linear) chains play a role in directing DNA repair or direct autophagy 3. Monoubiquination also plays traficking roles (Ex. Endocytosis) Different chains increase the diveristy of ubiquitin as a signal ubiquitin code

Answer 37

Proteins targeted for degradation have recognition motifs for E3 - Recognition motifs for E3 = degrons Example: 1. Motifs in substrate proteins - Ex - PEST sequences rich in P,E,S,T Amino Acids on cyclins (Cylcins may need to be phosphylated on residues to be recognized by machinery) - Ex 2 - Detsruction box (D Box/Ken Box) 2. N-terminal residues can be destabilizing and lead to protein degredation (called N-end rule substrates) 3. An exposed hydrophobic patch in misfodled proteins can act as a degron (Patch can bind E3 directly or indirectly through a chaparone bridge)

Answer 38

N-terminal residues can be destabilizing and lead to protein degradation Arg,Leu,Phe resiudes (IF at the N-temrinus) are recognized by particular E3 and promote degradation - Most proteins initiate with methionin --> the Arg,leu, Phe at the N terminus come from proteolysis the protein which exposes a new N terminal and reveals the amino acids (Piece of protein cleaved off is degraded) Example – happens in cohesion (holds SC together in mitosis) --> Separate of sister chromatids is due to degradation of cohesion with N-end rule

Answer 39

Used biochemistry to identify/isolate the components and find the mechansim by which proteins are tagged with ubiquitin and delivered to the proteosome They notices that protein degradation consumed ATP - Interested because biochemically the degrdation of protein shsould release energy BUT they found that the system was using energy (showed that ATP was used to activate ubiquitin and was used to unfold proteins to allow them to enter the proteosome)

Answer 40

1. Misfoloded protein degredation by UPS 2. Cell cycle regulation 3. Transcription factors 4. Antigen presenttaion 5. ERAD

Answer 41

An exposed hydrophobic surface = indictes of protein misfolding an serves as degredation signal When proteins fold they fold the hydrophobic regions the the inside of the protein so that the hydrophobic region is away from the aqaueous solvent ; WHEN proteins are misfolded they expose the hydrophobic patch

Answer 42

E3 recognizes the exposed hydrophobic regions Recognizes: 1. Recognize the hydrophobic regions directly 2. Indirectly through recognize of chaperones or other facts that bind to a unfolded regions - E3 sees when protein is bond to chaparone for a long time --> E3 knows this is a misfolded protein that needs to be degraded

Answer 43

Cyclins = key regulators of cell cycle transitions (oscillate during cell cycle) The sudden fall in cyclins = due to their degradation by the UPS

Answer 44

E3 that regulate the cell cycle can be complex with many subunits (Example – SCF E3 ligase) - SCF targets is first marked for degradation by phosphorylation - SCF E3 = has multiple SU = allows many oppertunities for fine tune regulation There are many SCF-type E3s (Each has a unique F-box protein ; F-box recognizes different targets) - Target = phosphorylated cyclin/other cell cycle proteins SCF E3 = brings E2 in close proximity with target = allows polyubiquination of the target and degradation of the target

Answer 45

2nd E3 in cell cycle = APC APC is more complex than SCF - ALL of the SU need to be properly assembled in order to have anaphase to metaphase transition APC = brings E2 close to substate = allows for degredation of the substarte through the proteosome

Answer 46

Transcription factors are regulated by VCB/HCL which is a SCF like E3 ligase Example - Hif1a - Hif1a is hydroxylated --> hydroxylation allows for Hif1a to bind to VHL E3 --> when bound to VHL E3 --> HIF1a is polyubiquinated and degraded Normal oxygen - Hif1a is made hydrozylated --> binds to VHL --> degrded Low oxygen - Hypoxia Hif1a is NOT hydrolxylated so it is not longer ubiquinated --> HIF1a goes to the nucleus and regulates a whole program of genes needed in hypoxia

Answer 47

Example TF regulated by UPS – NFkb (key TF in inflammatory response) Normally - Nfkb is sequestered in cytosol through association with inhibitor (IkB) Extracellular signal (ex TNFa) activates a signaling pathway --> get phosphorylation of IkB --> phosphorylated ikb is recognized by E3 and ubiquinated and degraded by the proteosome --> NOW Nfkb can enter the nucleus and turn on transcrioption of genes used in the inflamatory response

Answer 48

Overall - Use of proteome to put end products of protesome degredation to use by allowing for killing of virally infected cells Virally infected cells are killed by cytotoxic T cells - Infected cells are recognized because they present fragments of viral proteins on MHC 1 molecules on their cell surface that allows the T cell t recognize and kill the infected cells

Answer 49

MHC1 = in the lumen of ER BUT they can’t leave until they are bound to peptides Virally infected cells are factories for many viral proteins --> some of the proteins will misfold and are ubquinated and degtaded by the proteosome into peptodes --> peptides go to ER lumen where they bind to MHC1 molecules MHC1 bound to viral peptide can exit the ER and go through the sectrory pathway --> can be exposed on he cell surface to notify T cells to kill the cell

Answer 50

When virally infected cells makes interferon --> interferon upregulates several proteasome core beta SU (LMPs) Beta SU modify the favor the production of peptides that terminate after basic or hydrophobic amino acids (cleavage after basic/hydrophic amino acid optimizes binding to MHC1) This form of the proteasome (when SU have been replaced) = “immuno-proteasome”

Answer 51

Degradation of misfolded ER luminal or membrane proteins is important in diseases (Ex. Cystic fibrosis) Misfolded proteins can exist in organelles but proteosomes E1, E2, and E3s are present in the cytosol and nucelus only - In the lumen of ER or ER membrane a misfolded protein is seprated from E1, E2, E3 and proteosome by ER membrane

Answer 52

Properly folded proteins are included in transport vesicles that bud off the ER and go to the golgi and to the cell surface BUT misfolded proteins in ER lumen remain chaperone-bound are not packaged into transport vesicles to leave the ER/don't go to cell surface Binding of protein to a chaprone in recognizes --> chaperone-bound misfolded proteins are retro-translocated from the ER to cytosol --> During retrotranlocation membrane bound E2 and E3 ligases add ubiquitin onto the protein --> protein can be degraded by the proteosome - Proteins are ubiquitinated on the cytosolic face of the ER membrane - Remove glycotcic loops in ER Retrotranslocon = is a transmembrane E3 ligase (retrotranlocation and ubiquinaton are coordincated by 1 molecule)

Answer 53

ERAD is involved in many diseases ; Preventing destruction of the mutant protein can help treat disease Example - Cystic Fibrosis - CFTR = chloride chanel at the plasma membrane in lung cells that allows for proper mucus in the lungs (uses secretory pathway for exit at cell surface) - Most common CF disease allele (CFTRdF508) prevents correct CFTR folding --> CFTR is retrotranlation + poly ubiquination + proteosome degreded via ERAD --> no protein at the plasma membrane

Answer 54

Drugs promote CFTRdF508 folding block it degradation by ERAD + promoter trafficking + boosts activity at Plasma membrane Drugs: 1. Drugs that prevent misfolding/corrects folding of mutant = protein can exit from the ER and go through the secretory pathway and go to the plasma membrane - CTFR is still mutant at the plasma membrane 2. Second drug helps boost activity of the mutant protein at plasma membrane (NOW have mutant CFTR being able to secrete Cl again) NOW use 3 drugs - 2 chemical chaperones to fold the mutant protein well AND 1 drug boosts activity Drugs = help circumvent ERAD degredation pathway for thearpy

Answer 55

Many receptors need mono ubiquination as traficking signal in 2 steps of the endocytic pathway: 1. Monoubiquination allows for recruitment to the clathrin coated vesciles for endosome function 2. Receptors to be recruited via ESCRT machinery into ILVs of MVB that are degraded by the lysosome

Answer 56

Example - After signaling EGFR is monoubiquinated Have a E3 that recognizes EGF repcetors when it has been done signlaing for a while --> E3 puts mono ubiquitin on the C-temrinal tail --> Eps15 has ubiquitin interaction motif that reognized monoubiquitin on EGF receptor --> different domain of Eps15 recruits and binds binds to the adapter protein (AP2) --> adapter protein AP2 binds to clathrin --> clathrin intiates endocytosis END – monoubiquination (NO CHAIN) is a signal for endocytsisis NOT proteosome degredation

Answer 57

Monoubiqionatino allows the stimulation of EGF receptor for down regulation by singling to include the EGF receptor in clathrin coated vescile that will lead to endosome fomration and degredation/recylcing

Answer 58

Another novel role for monoubiquination in trafficking = providing a signal for incorporation into MVB (Cargo needs to be Monoubiquited to be sort/concetrated cargo into intraluminal vesciles of the MVB that allows for the protein to be degraded) Mono ubiquitin sorting tag promotes recognition by a UBD domain in a component of the ESCRT 1 complex

Answer 59

Ubiquitin provides a regulatable signal and is analogous to phosphorylation Ubiquitin conjugation machinery (E2/E3s) and Deubiquinases (DUBs) are analagous to kinases and phosphatases This (E2/E3 and DUBs) provides the cell with another versatile regulator in addition to phosphorlaytion for many processes

Answer 60

UBLs = Proteins are related to ubiquitin - Might or might not have a strong sequence relationship BUT they all have a strong folding relationship (all have a beta-grasp fold) UBL attachment to protein similar similar to ubiquitin - C-terminal Gly of the UBL is conjugated to a NH on a Lys on target protein - Enzymes that add UBL to protein are related to ubiquitin E1, E2, E3

Answer 61

Examples: 1. Sumo: nuclear functions (localization and gene regulation) 2. Nedd8/Rub1: stimulates SCF E3s 3. UCRP: regulates certain signal transduction pathways 4. Hub1: regulates cell polarity (yeast) 5. Apg12, 8: regulate autophagy 6. Urm1: function unknown

Answer 62

Activation of autophagy resembles activation of proteasome degradation (uses enzymes similar to E1 and E2) - Apg genes mediate formation of complexes that are important for intiating phagfore formation Apg12 gets conjugated to a target portein Apg5 (Important for intiating autophagy ) Apg8 (aka LC-3) is conjugtaed to a target lipid (PE) END - Apg12-Apg5 and Apg8-PE promote the formation of autophagic veciles (help intiate elongation of phgafore) - Most compenents are recycled BUT Some LC3 remains in the autophagasome

Answer 63

Can use GFP-LC3 as a marker for autophagic vesciles (GFP-LC3 is fused in cells) GFP-LC3 in no stravtion = is diffuse in cells (no puntate strcutures) Stravation have formation of many puncate strcutures (puntact structures = autophagasomes)

Answer 64

SUMO and ubiquitin both have beta grasp fold SUMO and ubiquitin are both conjugated to a substrate lysine by the carboxyl group of its C-terminal glycine Substrates can be mono- or multi- SUMOylated The SUMOylation machinery is strikingly similar to the ubiquitin E1/E2/E3 machinery But SUMO does not direct degradation INSTEAD it regulates function of proteins

Answer 65

SUMO modifies many nuclear proteins - In general SUMO turns off the function of the protein to which it is attached Example - modified proteins involved in regulation of transcription + chromatin structure + nuclear pore localization + DNA repair

Answer 66

1. UB and UBLs have diverse surfaces available for macromolecular interactions 2. Ability of cells to use structurally distinct chains or mono UB/UBL greatly expands their potential as signaling molecules (can be in chains that helps recruit other molecules) 3. Some target proteins can be modified at the same site by distinct Ub or UBL modifications which fine-tunes distinct functional fate Example - PCNA has distinct activities depending on its modification status at 1 site

Answer 67

PCNA has distinct activities depending on its modification status at 1 site - PCNA binds to DNA polymerases and influences the mode of DNA repair - PCNA can be modified at a specific site either by Ub or SUMO polyubiquination (K63 ubiquitin chain) = promotes error-free DNA repair SUMO prevents error free repair and promotes error-prone DNA repair

Answer 68

UPS participates in just about everything Example diseases: 1. HPV: virally-induced cervical cancer (viral protein [E6] causes an E3 [E6AP] to act on host p53) 2. Parkinson’s: neurodegenerative disease E3 [parkin] mutations 3. Angelman's syndrome: E3 [E6AP] mutations Many hereditory disease map to genes that encode E3 or E3 recognition site

Answer 69

Proteostasis = protein homeostasis Proteosasis = important in disease and aging Proteostasis declines as we age

Answer 70

Proteostasis depends on well-functioning degradative systems (UPS and lysosomal) Young = have good protein qulaity control systems (have misfolded proteins BUT they can be handled by quality control systems) Old = quality control systems decline = misfolded proteins accumulate = get misfolding disease Example - Hallmark of neurodegenerative diseases of aging is accumulation misfolded protein aggregates (Alzheirmers + Parkinsons + ALS + Huntingtons)

Answer 71

Interventions to increase proteostasis are being explored as anti-aging drugs Example - Rapamycin increases the longevity and healthspan in mice - Rapamycin regulates mTOR --> adding rapamycin increase autophagy which appears to alleviate the aging process