Protein Turnover: Degradation by Lysosome & UPS Flashcards
intracellular protein degradation is compartmentalized
-would be an issue if proteolytic enzymes were running awry in the cells
-degradative enzymes in compartments
proteasome
-protein-limited compartment
-interior of chamber where proteolytic enzymes are located
lysosome
-membrane-limited compartment
-filled with proteases, lipases and other hydrolases- kept acidic with proton pump
what are the many roles for protein degradation?
- allows cells to respond to changing conditions or physiological stress
- protein quality control: misfolded or damaged proteins can be harmful for the cell, so need to be degraded
- acute regulation- degradation can rapidly turn cellular processes on and off (faster than transcriptional regulation)
allows cells to respond to changing conditions or physiological stress
-starvation- cell needs to turnover all of its existing proteins –> create new building blocks
-hypoxia- number of oxygen-consuming enzymes in the cell- under conditions of low oxygen, cells need to get rid of those oxygen utilizing enzymes and synthesize new program of cellular components
-differentiation- cells want to get rid of whole programs of proteins at one stage in development to make way for another program
protein QC
-misfolded/damaged proteins being degraded- harmful for cell since they can retain part of their function such as binding to a cell surface receptor but not be able to carry out other parts of their job like downstream signalling
-can arise due to mistakes in folding as protein is being synthesized or synthesizing proteins from mutant gene
-physiological insults like oxidation and heat shock can unfold already folded proteins and these need to be disposed of
acute regulation
-can rapidly turn cellular processes on and off
-Ex. timed destruction of cyclins that moves cell cycle forward
how is protein degradation studied?
-pulse-chase- allows investigators to follow fate of small cohort of newly synthesized proteins
1. pulse label for a few minutes by addition of radioactive amino acid to culture medium that’s taken up
2. labelling is terminated by addition of excess of cold methionine that swamps out the label
3. at the end of the pulse, labeled small cohort of newly synthesized proteins
4. chases for limited amount of time by simply allowing growth shown on the x-axis
–> at each time point after the pulse, you would prepare a protein extract and IP the protein then run on gel
pulse-chase experiment with yeast strain
-included another yeast strain that’s a useful mutant with a deletion of protease PEP4, a master vacuolar protease (lysosome in mammalian cells)
-when you get rid of the protease, no other proteases can work in the yeast lysosome
-in the WT you can see the protein begin to disappear but in the deletion strain, it consistently appears
cycloheximide chase
- add protein synthesis inhibitor (most often cycloheximide)
- chase refers to allowing the cells to grow for increasign amounts of time in the presence of cycloheximide
- prepare protein extract and detect your POI with western blot
–> following the POI in WT and you see very little degradation –> stable protein
–> mutant POI and repeat experiment –> see the mutant is unfolded and rapidly degraded
to determine degradation pathway for unstable protein in mammalian cells, add inhibitors that block the lysosome or proteasome
-lysosomal proteases have low pH optimum –> if you raise the pH of a lysosome, you can block proteasomal degradation
-drug commonly used to interfere with lysosomal acidification is bafilomysin A, which inhibits the lysosomal ATPase and lysosome becomes less acidic
-proteasome- specific drugs like MG-132 and bortezomib bind to and block protease active sites in the proteasome
multiple pathways for entry into lysosomes
- endocytosis
- autophagy
- chaperone-mediated autophagy (CMA)
endocytosis
cell surface proteins can be incorporated into clathrin-coated vesicles that fuse to form early endosome that matures into late endosome –> fuses with vacuole and all of the contents of the endosome are degraded
EGFR
-receptor tyrosine kinase
-when its ligand EGF binds to the receptor, that allows this to auto-phosphorylate and become an active signaling molecule at its cytosolic tail and turns on a number of downstream pathways
-cell will eventually want to turn this signal off and use its receptor down regulation
-involves inclusion of these activated receptors in clathrin-coated vesicles and clathrin falls off
-these fuse to form early endosome and delivered to the lysosome for degradation
-cytosolic signaling part of the EGF receptor is maintained since it’s still facing the cytosol
formation of intraluminal vesicles- once inside these, signaling is terminated and now this whole molecule, once the MVB fuses with the lysosome, is available for degradation
-this type of budding event is budding away from the cytosol into the lumen
-clathrin coats deform vesicle, the membrane, and form endosomal vesicles and clathrin is recycled
ESCRT complexes mediate ILV formation
-components are called VPS genes and conserved in mammalian cells and organized into complexes
-monoubiquitin- important signal that signals that this protein should be included in these intraluminal vesicles
ESCRT proteins don’t surround ILV –> promote inward vesicle formation
complex sits at the surface of endosome and forms spiral-like structure that pushes down a vesicle into the interior of the endosome until it’s fully formed
retroviruses co-opt ESCRTs for their own purpose
-budding out of the cell
-we have the ESCRT complex forming ILVs- process starts with Vps27 or HRS, which sits at the surface of the lysosome and recruits ESCRT machinery
-machinery can be co-opted by retroviruses like HIV, which encodes Gag protein that sits at the surface of the PM and recruits ESCRT complexes to that site and allows budding of viral particles
autophagy
-lysosomes are able to degrade bulk amounts of cytoplasmic molecules that have been packaged into autophagosomes
-allows cells to break down obsolete parts of itself for disposal or reuse
-start off by formation of double membrane structure called phagophore ad source of double membrane is ER membrane and whole chunk of cytoplasm is included in the growing phagophore and completes growth to become autophagosome
-autophagosome fuses with lysosome and contents are degraded
steps in autophagy
starvation signal –> initiates phagophore formation that becomes autophagosome then docks with lysosome and allows degradation
dramatic progress in IDing components of autophagy
-ID components was carried aout by looking at yeast genetic screen that IDed 35 ATG (APG) genes
-autophagy has been linked to neurodegernation, aging, etc.
-some evidence suggests it promotes or hinders cancer
-mTOR- master kinase- sits on the surface of lysosomal membrane and senses nutrient levels in the cell
selective autophagy
-autophagy can be selective and remove specifically damaged organelles, protein aggregates, and pathogens
-these organelles expose signal to make them cargo molecules and interact with phagophore-forming structures
autophagosome and ILVs from the MVB have the same fate –> lysosomal degradation
very different in terms of machinery –> double membrane in autophagy and single membrane with endosomes but they all fuse with lysosome for degradation
chaperone-mediated autophagy
-another starvation pathway
-proteins enter lysosome by non-vesicular pathway –> uses chaperones and transport channel on the lysosome to enter the lysosome
-proteins with a recognition sequence (KFERQ signal)- 20% of all proteins have something that can be recognized by KFERQ signal
-recognized by cytosolic membranes of the heat shock family and such proteins are directed to the lysosome and reeled in to lysosome by a chaperone protein of the Hsc70 family in the interior of the lysosome
CMA steps
KFERQ proteins bind to Hsc70 chaperones and co-chaperones –> allows delivery to lysosomal channel protein called Lamp-2A –> when there’s cargo bound to it, this multimerizes and forms channel
Ubiquitin-Proteasome System (UPS)
-protein tag Ubiquitin is covalently attached to lysine residue in a target protein by series of enzymes (E1, E2, and E3)
-after a single ubiquitin is added here, additional ones can be added to form a chain
-ubiquitin chains (>4) are recognized by proteasome and the target protein with such a chain is now degraded into small peptides
how do you experimentally determine whether a POI is degraded by the UPS?
-IP POI and blot with anti-ubiquitin antibodies and if the POI has one lysine, you can see defined ladder
-if you add a proteasome inhibitor, you’ll see that b/c degradation is being blocked, longer and darker chains form
-difficult to detect ubiquitinated proteins since they’re transient intermediates due to their rapid degradation
-you often see high mw ubiquitin smear and if you add a proteasome smear, it gets dark –> very heterogeneous group of proteins and that’s because proteins have many lysines
-E1- ubiquitin-activating enzyme and ATP is hydrolyzed to activate ubiquitin, which is covalently bound to E1 in thioester linkage –> passes ubiquitin to E2 conjugating enzyme, which transfers ubiquitin onto a target protein and E3s are called ubiquitin ligases, which are the specificity factor of the system
26S proteasome
-large structure
-proteasome degrades proteins into peptides, usually 7 AAs or smaller
-2 major components: 20S core (where proteolytic enzymes are located) and 19S cap (number of regulatory roles)
20S core-proteolysis
-proteases in the proteaome core need to be sequestered away from the cytosol –> active sites inside the chamber
-protein is being degraded as it passes through the chamber
-stack of 4 7-membered rings with alpha subunits and beta subunits have proteolytic activity
-beta subunits each have specific proteolytic activities- cleave after acidic, basic, or hydrophobic residues
proteasome inhibitors bind to beta subunits of 20S core and block activity
-Mg-132 and bortezomib bind inside proteasome channel to active sites of the beta subunits
-pore of 20S core is very narrow
19S proteasome cap- regulates substrate entry into the 20S core
-cleaves ubiquitin off substrate protein before it enters the core –> allows ubiquitin to be recycled into the cell
-ubiquitin receptor protein- helps recognize ubiquitin chains >4
-unfoldase ring comprised of 6 triple A ATPases that form unfoldase ring and use energy of ATP hydrolysis to unfold target protein
-ubiquitin hydrolase- cleaves ubiquitin off protein as it’s being unfolded
ubiquitin
-small protein and gets linked to lysine on the target substrate
-beta-grasp fold
-main polypeptide backbone with ubiquitin side chain
-multiple ubiquitins can be linked to substrate
-glysine carboxylate is linked to a lysine in the target protein and 2nd ubiquitin can be added to the first at the particular lysine within ubiquitin itself
-carboxyl group of second ubiquitin is forming peptide bond with lysine 48 in ubiquitin and process is repeated
2 major classes of E3s
- HECT E3s- as Ub is being transferred from the E2 onto the target protein, HECT E3s form transient covalent intermediates with Ub
- RING E3s- majority of E3s and they do not form covalent intermediates with Ub- they bring E2 close enough proximity to target to allow transfer to occur
UPS
-Ub is activated by being linked to an E1-activating enzyme that transfers Ub to an E2-conjugating enzyme
-E3 Ub ligase brings in E2 in close proximity with substrate and allows trasnfer of one Ub onto the lysine of the substrate
-Ub chain forms and proteasome cap can regonize a chain of >4 Ubs in the K48 linkage
-cap cleaves off the Ub and unfolds the protein to be degraded into peptides
proteins targeted for degradation have recognition motifs for E3s called “degrons”
- motifs in proteins like cyclins have PEST sequences and in some cases they may be phosphorylated on these residues or destruction boxes
- N-terminal residue- can be destabilizing and lead to proteasomal degradation- N-end rule is if you have Arginine, Leucine, or Phe at your amino terminus, you will be rapidly degraded
- exposed hydrophobic patch in misfolded proteins
cell cycle regulation
-cell cycles oscillate- cyclins A and B you see a lot then they decline then you see a lot and they decline
-degradation was occurring by UPS
-E3s that regulate the cell cycle can be very complex with many subunits
Ex. SCF E3 ligase that differ by their F-box (region that recognizes the target) –> you can see that it brings E2 in close proximity with the target and allow for polyubiquitination
the abundance of some transcription factors like HIF-1 alpha can also be regulated by SCF-like E3s
-HIF-1 alpha works to regulate gene expression in low oxygen
-HIF-1 alpha is hydroxylated and allows it to bind to E3 VHL- once it’s bound, it’s polyubiquitinated and degraded
-under normoxia, HIF-1 alpha is constantly being made and degraded but when cells experience low oxygen, HIF-1 alpha can no longer be hydroxylated and no longer ubiqutinated –> can go into nucleus and upregulate a whole program of genes that are needed for oxygen
TF- NF kappa B
-regulates inflammatory responses- TF that’s a heterodimer composed of these 2 subunits and bound by inhibitor IKB
-when there’s extracellular factor, it can activate a signalling pathway that now phosphorylates IKB
-when IKB is phosphorylated, it is recognized by an E3 and now is polyubiquitinated and degraded
-NF-KB goes into the nucleus and turns on inflammatory response
antigen presentation
-virally infected cells have MHC-I molecules bound to viral peptides onto the cell surface that are recognized by T cells
-peptides are made when virally infected cells become factories- some proteins misfold and are degraded by the proteasome into peptides
-these are transported into the lumen of the ER and they meet up with MHC Class I cells –> MHC Class I molecules bound to viral peptides can now exit the ER and go through the secretory pathway to the cell surface
-interferon upregulates a umber of beta core subunits that favor the production of peptides that terminate after basic or hydrophobic amino acids that optimize binding to MHC Class I
ER-associated degradation (ERAD)
-misfolded proteins can exist in all compartments but the proteasome and Ub apparatus are present only in the cytosol and nucleus
-in the ER lumen, misfolded protein is separated from the proteasome by ER membrane
-if you have properly folded protein, it can be included in the transport vesicles that bud off the ER -> golgi -> cell surface
-if protein is misfolded in the ER lumen, it’s bound to a chaperone and not allowed to be a cargo molecule in these transport vesicles
-misfolded ER luminal protein bound to chaperone and can’t get out to cell surface –> ends up being retrotranslocated out of ER lumen
-there are E2 and E3 ligases that added Ub onto the protein as it’s being retrotranslocated
-there’s also glycolysis in the cytosol that cleave off glycolysol groups that were put onto this protein in the ER and now protein can be degraded by proteasome
non-traditional use Ub: mono-ubiquitination of some receptors tags them for endocytosis
-after signaling, EGF receptor undergoes monoubiqutination- particular E3 that recognizes EGF receptor when it’s undergoing prolonged signalling and puts monoubiquitin at different places on the C-terminal tail and is recognized by Eps15 protein that has Ub interaction motif and is able to bind adaptor proteins that interact with clathrin –> monoubiquitin is allowing the stimulated EGF receptor for down regulation to be included in clathrin-coated vesicles that lead to endosome formation
-cargo molecules need to be monoubiquitinated to be incorporated into interluminal vesicles
ubiquitination provides a regulatable signal analogous to phosphorylation
Ub conjugating machinery and deubiquintinases are analogous to kinases and phosphatases that provide cells with versatile regulators
activation of autophagy resembles the activation of proteasomal degradation
-Ub pathway with E1, E2, and E3 forming polyubiquitn chains
-APG12 gets conjugated to a very specific target APG5
-APG8, also called LC-3, is also conjugated to target lipid and various APG genes mediate formation of these complexes
-important for initiating formation of phagophores- Apg12-5 complex and Apg8-PE complex help to initiate the elongation of phagophore
SUMO-like Ub
-beta grasp fold and is conjugated to the substrate lyssines at the C-terminal glycine
-substrates can be mono or multi-sumolyated and machinery is similar to Ub machinery
-SUMO doesn’t direct the degradation of proteins but regulates function of proteins and has many nuclear roles
what features make Ub and UBLs such great signals for regulating the activity/localization of their targets?
-as proteins, they ahve diverse surfaces for macromolecular interactions and can be in chains to help recruitment of other molecules
-some target proteins can be modified at the very same site by either a distinct Ub or UBL to fine-tune its fate
PCNA has distinct activities depending on its modification status at a single site
-protein trimer that binds to DNA polymerases and influences DNA repair
-can be modified at a specific site either by Ub or SUMO
-K63 chain promotes error-free DNA repair, whereas SUMO prevents this mechanism and allows error-prone DNA repair to occur
proteostasis (protein homeostasis) appears to decline as we age
QC systems decline as we age and protein misfoldings accumulate
