Lecture #9 - Protein Turnover Flashcards
Why is intracellular protein degredation compartementalized
Would be an issue if proteolytic enzymes are all over in cytoplasm –> instead degrative enzymes are sequestered into compartments
Compartments:
1. Protein limited (protesoomes that contain proteolytic enzymes)
2. Membrane Limited (lysosomes filled with proteases + lipases + dryloases)
Acess into to the compartments needs to be tightly regulated
Optimal pH of lysosom enzymes
ALL have acidic pH optimum
Lysomses if kept acidic by proton pump
Roles for protein degredation in cell
- Degredation allows cells to repond to chnaging conditions or physiological stresssed
- Ex - starvation causes cells to trunover all existing proteins to make new building blocks so new proteins can deal with stravtion
- Ex 2 - Hypoxia -Get rid of oxygen consuming enzymes and synthesize new program of cellular compoennts
- Ex 3 - Differentiation
- Protein quality conrtol
- Protein degradation can accomplish acute regulation
Faculty degradation is the cause of many disease
Protein turnover in Differentiation
Cells/tisues wnat to get rid of whole programs of proteins at once stage in dvelopment to make way for a whole new program of proteins
Example – In erythropeisis hematopetic stem cells differentiate into mature RBCs
Protein quality control
Protein quality control = misfolded proteins being degraded
Misfodled/damaged proteins can be harmful to the cell (need to be dgeraded)
- Misfolded proteins can have half function which leads to dominant negative effects (ex. Can bind to the receptor BUT can’t do downstream signaling )
How do misfolded proteins arise
Misfodling can arise due to mistakes in folding of newly synthesized proteins or due to syntheszing proteins from a mutant gene
Folded proteins can be unfolded because of physiological (Ex. oxidation or heat shock
Role of protein degredation in cell - Regulation
Acute regulation - degradation can rapidly turn cellular processes on/off (can turn things on/off faster than transcriptional regulation)
Example 1 - Cell cycle progression (time destruction of cyclins s the cell cycle can move forward)
Example 2 - After responding to extracellular signals cells can terminate the response
How is protein degradation studied
Overall – use pulse chase analysis to follow the fate of a small cohort of newly synthesized protein
Pulse chase - Process
- Pulse label cellular proetins for a few minites by adding radioactive Amino Acid (S-methione) to medium to be taken up by the cells
- After pulpse terminate labeling by addition of excess of unlabled “cold” methionin (swamps out the label)
- NOW have labeled a small cohort of newly synthesized proteins
- Chase by allowing cells to grow for increasing lengths of time
- At each time point after pulse prepare protein extract and immunoprecipiatte the protein of interest and run gel and could bands by autoradography
Graph from pulse chase
Graph shows half life
Half life = times it takes for 50% of the labeled molecules to be degraded (disappear)
Pulse chase reuslts:
Gel – After pulse chase labeling of cellular proteins (proteins are HA tagged Step6)
- Protein was Immunoprecipiated with anti HA antibodies
Used yeast strain (mutant with deleted Pep4 protease)
- Pep4p =master vacuolar protease (remove Pep4 protease –> no protease works in yeast lysosome)
Results - When delete Pep4 then the protein is stabilized
- Because protein is stable when remove Pep4 = means the protein is degraded in the vacuole
Vacouloe in yeast
Lysosome = vacule in yeast cells
Pep4p =master vacuolar protease
How is protein degradation studied now
Instead of pulse chase use cyclohexamide chase
- Chase = allowing cels to grow for increasing amount of time
- Prepare protein with Western instead
Process:
1. Add protein synthesis inhibitor (often inhibitor is cylohexamide) in cells so no new protein is made
2. Chase by allowing cell growth for increasing lengths of time in the presence of cyclohexamide
3. Prepare protein extract at each time point and detcet your favorite protein (YFP) by western blot
Cyclohexamide chase - results
Gel:
- WT – over time there is no degradation of the protein (protein is stable)
- Mutant – Mutant version of the protein is rapidly degraded (Suggest that the protein is unfolded and degraded)
- Can see the half life
How to detemrine if a protein is degraded by the lysosome or the proteosome
To determine the degradation pathway –> use chemical inhibitors that block the lysosome or the proteosome can be included in the chase
To inhibit lysosoem - becuase lysosomal proteases have a low pH optimum if you raise the pH of the lysomes you block protein degredation
- Raise pH with Weak bases (Ex. Chloroquine or NH4Cl) OR drugs that interefere with lysomal acidifcation (Ex. Bafilomycin A1 –> inhibits the the lysomal H+ ATPase)
To inhibit the proteosome - Use proteosome-specific inhbitor drugs (Ex, MG132 Bortezomib which binds to and blocks the protease active sites in the proteosome)
Issue in cylohexamide chase
Cytohexamide chase is NOT as robust as normal pulse chase
Pulse chase is non-evasive (not changing the cells) Vs. In cylohexamide chase you block all protein synthesis which can have secondary effects
Pathways for entry into lysosomes
- Endocytosis
- Autophagy
- Chaparone-mediated autophagy (CMA)
Endocytosis
Overall – cell surface proteins are internalized into a clathrin coated vescile–> veciles travels and fuses to form the early endosome –> early endosome matures to the the late endosome (MVB) –> late endosome fuses with the vacule and all the contents of the endosome are degraded (go to the lysosome to be degraded)
Example of endocytosis
EGFR
Ligand binds to the EGF receptor –> binding cuases autophosphorylation of the receptor –> allows the receptor to be signaliong molecule at the cytosolic tail –> truns on downstream pathways –> eventually the cell needs to terminate the signal and uses recetor down regulation to terminate the signal
Down regulation = internalization of activated receptor in clathrin vescile –> Clathrin falls off –> vesicle fuses to form the early endosome –> ultimately get lysomal degredation
What hapepsn between the early endosme and the lysosome in downregulation of EGFR
Overall - internilized recpetpors are incorpoated into multi-vescilualr body (MVB)
MVB = late endosome
Why is incorpoatig receptor into MVB important in terminating the signal
Protein keeps topology over the course of membrane traficking –> cytoslic part of teh EGF receptor stays in teh cytoplasm after internilization AND in the early endomses = receptor can still do siganing because it is still facing the cytosol
IF the early endosome (with the receptor) fused to the lysomes the signaling end would still be sticking out and would not be degraded and could continue signlaing
Solution – form an intraluminal vescile (MVB) –> once receptor is inside the intraluminal vescile the signling is termoniated
- Once MVB fuses with the lysosme the whole molecule of the receptor is able to be degraded
Budding in MVB
IN MVB the budding is budding away from the cytosol into the lumen of the endosome
Different from other budding:
- Example - When have clathrin coats they defrom the membrane and form endosomal veciles and then clathrin falls off and is recyled VS. In MVB if defomed the vescile with clathrin coat then once fused with the lysosme the coat protein would be degraded
Solution there is a ESCRT complex is used instead of coat
How does the MVB form?
The early endosome matures to became an MVB when intralumenal vesicles are pinched off the membrane into the interior –> signaling stops when receptor is in the ILV
THEN The MVB fuses with the lysosome –> ILVs and the proteins they contain are degraded
ILV allow for the enter protein to be deraded Vs. no ILV the lysosomal hydrolases would not have access to the entire molecule and would therefore not effectively degrade and inactivate
ESCRT complex
Function - mediate Intraluminal Vesciles
ESCRT compoenets are organized into complexes:
- ESCRT 1 - Recognzes cargo and begins the process of intraluminal vescile formation
- ESCRT2/3 Finsihes the formation of the vescile
Overall – ESCRT 1 Complex recgiznies –> ESCRT 2,3 complex assmebly –> Cargo/sorting vescile formation –> diassmebly and Recycing of MVB sorting components
What mediates intraluminal vescile
Monounbiquitin of cargo mediates its sorting into intraluminal vesciles
- Monoubiquination signlas that the protein should be included in inraluminal vescile
What do ESCRT complexes promote
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
ESCRT proteins Vs. Coat proteins
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)
Retroviruses and Coat proteins
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
Autophagy
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
Image of autophgasome
Image = autophagasome in EM (see the double membrane srutcuere)
Steps in Autophagy
- Starvation signal that intitaes the phagafore formation (double membrane stcuture)
- 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
- Autophagosome fuses with the lysosome allowing for degradation
- Lysosomal hydrolases break down the autophagic body –> allowing recycling of molecular components
Idetofying components in autophagy
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
mTOR and autophagy
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
What is autophagy involoved in
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
Selective Autophagy
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
How does selective autophagy work
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
Fate of Autophagasomes and ILVs
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
Chaparone mediated Autophagy
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
How do proteins get to lyssome in CMA
Uses KFERQ recognition motif
- 20% of protein have something that can be recognized as KFERQ
- 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 - Protein unfolds and passes through Lamp-2A transport channel inot the lysosome by a Hsc70 chaparone inside of the lysosme
- 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
Overview of uniquitin proteosome system
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
What is often seen when detcting ubiqinuated proteins
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
UPS E3
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
HECT E3s
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
RING E3s
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
26S proteosome
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
20S core
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
Proteosome inhibitors
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)
Pore of 20S core
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
19S protosome cap
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
Ubiquitin Link to protein
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
Steps of Ubiquitin proteosome system
- Ub is “activated” by covalent binding of the E1 ubiquitin activating enzyme
- Ub is transferred to an E2 ubiquitin conjugating enzyme
- 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
- 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)
- 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
- The substrate protein is degraded and Ub is recycled
Types of chains made by Ubiqutin
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
Examples of cellular processes that Utilize UPS - Misfoloded protein degredation by UPS
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
Examples of cellular processes that Utilize UPS - Cell Cycle Regulation
Cyclins = key regulators of cell cycle transitions (oscillate during cell cycle)
The sudden fall in cyclins = due to their degradation by the UPS
2nd E3 in Cell cycle
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
Regulation of the abundance of TF
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
Examples of cellular processes that Utilize UPS - Transcription factors
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
Proteosome beta SU during viral infection
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”
ERAD process
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)
Drugs for Cystsic Fibrosis
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
Two roles for monoubiquination in trafficking to lysosome for degredation
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
Monoubiquonation
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
2nd role of monoubiquination in trafficking
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
Activation of Autophagy
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
Use of LC3
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)
SUMO Vs. Ubiquitin
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
PCNA
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
What does UPS particpate in
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
Proteostasisis and anto aging
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
