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31 Cards in this Set
- Front
- Back
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We study two categories of extracellular proteases.
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Extracorporeal and intracorporeal
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Intracellular intracorporeal proteases
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cytoplasm, proteasome, lysosome, peroxisome
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What is the purpose of endogenous protein degradation? ~3
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Maintain free AA pool
regulate metabolic rxn, cellular processes discard defective proteins or invader things we don't want |
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Maintaining aa pool
What giveth? What taketh away? |
Dietary, aa synthesis, endogenous protein degradation* (biggest contributor)
Protein synthesis, aa to urea and carbon skeletons, aa to other things (e.g. histamine) |
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Big picture: What are ways we regulate metabolic rxn? 7
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interconvertible enzymes (covalently modify them)
allosteric effectors enzyme compartmentalization gene expression protein synthesis protein degradation (slow but unequivocal) |
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What ways did he mention that we regulate cellular processes by degradation: 3
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Cell cycle progression- requires proteins with cyclin destructive boxes (9 AA that undergo ubiquitination)
Cell surface receptors (internalization, recycling, degradation) Antigen presentation (presentation, degradation) |
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When do we discard defective proteins?
What are we using to do this? |
Too many copies, damaged proteins (ROS), nonsense proteins
Endogenous protein degradation |
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How do we destroy engulfed material?
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All ends up in lysosome
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Why do some proteins have half lives of years and some are minutes?
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Managed through sequences
long half-lives: small hydrophobic or hydroxyl-containing resudes at N terminal short half-lives: large hydrophobic and hydrophilic; PEST sequences on interior of molecule |
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Endogenous protein degradation:
Ubiquitin-proteasome System Purpose Process |
Housekeeping function. Degrades any unwanted proteins using 26S proteasome.
Also regulates intracellular signaling. Poly-ubiquitinate target protein and 26S proteasome eats. |
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What puts ubiquitin marker on target proteins?
What takes them off? |
Enzymes E1 E2 and E3.
E1: activating 1 E2: conjugating 30-40 E3: ubiquitin ligases (specificity) 100+ DUB: Deubiquitinating enzymes |
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What to know about Ub?
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Compact globular protein, heat-stable, ubiquitous in eukaryotic cells
At C terminus is a Gly (G^76) and at 48 there's a K (K^48). |
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How is Ub attached to target protein?
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E1 activates Ub by attaching Ub's Gly76 to its Cys residue via thioester bond (reactive). Uses an ATP.
E2 (conjugating or hopping enzyme): E2 has a Cys that takes the Ub from E1 (some choice here since many E2's) E3 (Ub-protein ligase) brings E2 and the target protein together, putting Ub on a Lys of target. |
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Where/how are additional Ub's added?
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An isopeptide bond is formed using the amino group of K48 of first Ub and the Gly C-terminal of next Ub.
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What happens if you use a different Lys on Ub (not K48)?
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Instead of going to the proteosome, the targeted protein will go to lysosome or something else
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What makes up the 26S Proteasome?
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2 caps of 19S Regulatory particle: need ATP to wind proteins into them
20S Core Particle: stacked rings of proteins, Thr protease |
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How does the 26S Proteasome function?
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-Recognizes Ub targeted protein
-open cap/lid (ATP dependent) -Removes Ub tags (DUB) -Unfolds target protein (ATP dependent), threads protein into proteolysis chamber -Proteolysis generates 8-10 residue fragments |
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What is the specific cleavage of 26S proteasome?
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3 Specific types
Chymotrypsin-like cleavage: C side of large hydrophobics and aromatics Trypsin-like cleavage: C side of basic residues Peptidyl-glutamyl peptide-like activity: C side of acidic and branched neutral (Val, Ile, Leu) |
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What is Ubiquitin-proteasome system's role in cell signalling?
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I-kB (I-kappaB) interacts with NF-kB to hold inert.
When stress, infection, cytokines: I-KB gets phosphorylated -> ubiquitinated, liberating NF-kB. NF-kB (proteins p50 and p65) goes to nucleus and induces txn |
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Ubiquitin-Proteasome Dysfunction:
Gain-of-function mutation example What is targeted and why is it important? How is it destroyed in which types? What is the treatment? |
HPV Human papilloma virus -> cervical cancer
Normal surveillance is by p53 tumor suppressor protein (looks for damage, gets phosph. and goes to gene to induce cell death) In types 16 and 18, HPV promotes Ub of p53 via E6 oncoprotein binds with E6-associated protein which makes an E3 ligase that targets p53 Velcade: targets 26S proteasome's Thr |
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Burkitt's Lymphoma
What does it affect? |
Affects: facial bones, cecum, ileum, ovaries/breasts, kidneys
c-Myc oncoprotein is txn factor for 15% genes- mutation inhibits its ubiquitination Drugs and surgery can usually treat |
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SUMO
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small ubiquitin-like modifier protein..
not involved in protein degradation but other things |
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What are other cytosolic proteases that can degrade proteins if lacking a Lys?
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Calpains
Caspases Tripeptidyl peptidase II (TPPII) |
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Calpains
How many? Dependent on? Inhibitor? How many proteins degraded by this? Calpain disease |
15+.
Ca2+ dependent (must be bound) Natural inhibitor: calpistatin 5% calpain 3 mutation affects titin (spring in muscle). Causes Limb-Gridle Muscular Dystrophy Type 2A (LGMD 2A) |
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Organelle-based Protein Degradation
Enzyme type? Environment? How do proteins get marked for it? |
Lysosome
Many: Ser, Cys, Asp- activated in the low pH Very acidic with ATP dependent proton pump Phagocytosis, endocytosis, autophagy, **LAMP2A chaperone |
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What is the protease of lysosomes?
Present at all ____ of the endocytic pathway Most are what type? What is its function? |
cathepsins
stages endopeptidases Bulk degradation, process antigens, proproteins, prohormones |
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Peroxisomes
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usually seen in biosnythesis and degradation of lipids
D-amino acids are degraded here (e.g. from cell walls of bacteria) |
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Ectoproteases
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Secreted out to extracellular but within the body (intracorporeal) proteases
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Angiotensin-converting enzyme ACE
What is it? Where does it come from? What does it do? |
Zinc metalloproteinase (carboxydipeptidase)
Lungs and kidney's blood vessels Angiotensin I to angiotensin II (constricts blood vessels increasing blood pressure) |
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elastase 2
What is it? Where does it come from What does it do? |
Neutrophil elastase hNE
Ser protease secreted by activated neutrophils and macrophages during inflammation Hydrolyzes proteins within neutrophil lysosomes (azurophil granules) and in bacteria |
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Plasmin
What is it? What type of enzyme is it? Where is it made/as what? |
clot-dissolving factor
an ectoprotease, Ser protease Liver secretes as plasminogen Binds fibrin and is activated by "tissue plasminogen activator" -> degrades fibrin clot for healing |
