30 - Mechanisms of Protein Catabolism Flashcards
Lysosomal degradation is non-selective.
How does it work?
How does it change with diabetes?
Lysosomes contain cathepsins, which are proteases which acidic optima
- Degrade proteins in endosomes and autophagic vacuoles
- Inactivated by chloroquine (antimalarial agent), increases pH inside lysosome.
Lysosome is not involved in the degradation of short lived normal proteins or abnormal proteins.
In diabetes, there is an increase in lysosomal degradation of proteins to meet the nutritional requirements of the cell.
What is proteasomal degradation?
ATP dependent protein degradation system that requires ubiquitin.
Covalent addition of ubiquitin to the target protein is energy dependent and marks it for degradation by the proteasome
Ubiquitin conserved among eukaryotes (not present in prokaryotes).
Give the three steps for the conjugation of ubiquitin to a protein
- Terminal carboxyl of ubiqutin is conjugated to E1 protein via thioester linkage and catalyzed by ubiquitin activating enzyme.
- Transfer of thioester to an E2 protein (ubiquitin-conjugating enzyme)
- Transfer of the activated ubiquitin from E2 to an ε-amino group of a lysine residue in the target protein forming an isopeptide bond (catalysed by an E3, ubiquitin protein ligase)
Describe the specificity of E proteins in proteasomes?
E1: 1 per organism (general)
E2: 10-20 per organism
E3: many (specific!)
E3s likely serve to determine the specificity of ubiquitination for a set of proteins
How are most proteins tagged for proteasomal degradation?
Polyubiquitination, where lys-48 of ubiquitin is linked in isopeptide linage to the C-terminal carboxyl group of the next ubiqutin
These can reach lengths of 50 or more ubiquitin residues
Ubiquitinated proteins are degraded to short peptides by a large cytosolic complex called the proteasome.
7-9 residue peptides are released and hydrolysed to amino acids in the cytosol
What are the three main components of the 26S proteasome?
- 20S cylindrical core
- 2 19S regulatory caps
Proteolysis occurs inside the 20S core and is extensive and processive. 19S identifies and unfolds the target protein
Ubiquitin chains are not degraded by the proteasome, they are removed and returned by enzymes associated with the 19S cap
How are proteins selected for proteasomal degradation?
- N-end rule (N terminal amin oacid of the protein determines how rapidly it is degraded
- Only explains degradation of proteins targeted by RING finger E3 (E3α), which targets the destabilizing amino acids. - PEST sequences (Pro, Glu, Ser, Thr rich sequences). Phosphorylation in this region targets protein for ubiquitination
- Cyclins are involved in cell cycle progression. Contain a 9-residue consensus sequence RTALGDIGN (destruction box) that is recognized by specific E3s
What are the three main components of the 26S proteasome?
- 20S cylindrical core
- 2 19S regulatory caps with AAA-ATPases
Proteolysis occurs inside the 20S core and is extensive and processive. 19S identifies and unfolds the target protein
Ubiquitin chains are not degraded by the proteasome, they are removed and returned by enzymes associated with the 19S cap
What does the 20S central channel of the proteasome insure?
Only entrance of proteins in unfolded (linear) conformation
- Active sites for proteolytic activities are located on inner surfaces of beta subunits
Several catalytic activities present (eg. trypsin like, chymotrypsin like, and others)
What are the AAA-ATPases associated with the 19S cap of proteasomes responsible for?
Recognition of ubiquitin and unfolding target proteins. Also for causing 20S conformational changes that open the end of the proteolytic channel.
What are deubiquitinating enzymes (DUBs)?
DUBs can release entire polyubiquitinated chain or individual units and may act as a timer for the degradation process. If protein is trimmed to less than 4 units, it may escape degradation.
Rpn11 subunit of 19S cap is responsible for removal of polyubiquitin chains before destruction.
Give 6 features of apoptotic cells
- Shrinking
- Condensation of chromatin at periphery
- Collapse of cytoskeleton
- Dissolution of nuclear envelope
- DNA fragmentation
- Blebbing of plasma membrane (due to loss of phospholipid asymmetry)
What are caspases?
What are the two classes?
Cysteine proteases with active site cysteine
- AKA cysteinyl aspartate-specific proteases
- Heterotetramers of large alpha and small beta subunits
- Produced as procaspases (zymogens) and cleaved to active forms by a caspase (autocatalytic)
Two classes: initiator and effector
What are caspases role in apoptosis?
Initiator caspases
- 2 death effector domains (DED) in a large prodomain
- Bind to target adaptor proteins by DED
Effector caspases
- Short prodomains
- Activated by initiator caspases
- Cleave a wide variety of proteins to cause apoptosis (cytoskeletal lamin, actin; cell cycle proteins; DNA replication proteins; signal transduction proteins eg. PKC).
Caspases 3 and 7 degrade a CAD inhibitor to activate DNAse (CAD). CAD cleaves DNA between nucleosomes to 200 bp DNA ladder increments
May also be to prevent transformation by exogenous DNA elements.
How does the extrinsic apoptotic pathway work?
The extrinsic pathway is the signal from outside to undergo apoptosis
- Eg. immune system induced via death receptor (Fas) binding to death receptor ligand (FasL)
- FasL binding causes recruitement of 3 receptor molecules which trimerize via death domains. Also recruits adaptors (binds to death domain of Fas) and initiator procaspase-8
- Cleaved caspase 8 activates and cleaves effector caspase-3, to activate it and downstream events in apoptosis.
