Intracellular Proteolysis Flashcards
How can protein activity be regulated?
- Active site and allosteric regulators
- Pro-proteins and zymogens
- Phosphorylation
- pH and protein degradation
How quickly are abnormal variants turned over?
Quickly (more quickly than normal proteins)
When do cells increase rate of protein turnover?
- Times of starvation
- Breakdown of muscle protein
What is the N-terminal rule?
Half-life of a protein is dependent on which amino acid on the N-terminal
How was the N-terminal rule tested for?
By mutating the N-terminal residue of Beta-galactosidase and half life is observed
What are the features of caspases?
- Cysteine dependent aspartate-directed proteases
- Mediators of apoptosis
- Regulated post-trans via pro-enzyme activation
What are the features of cathepsins?
- Major lysosomal protease family
- Cysteine proteases optimally active at low pH
What are the features of calpains?
- Ca2+ dependent non-lysosomal proteases
- Res
What are the features of pro-protein convertases?
- Ca2+ dependent serine proteases
- Cleave various proteins (e.g hormones and neurotransmitters)
- Activated by growth factors
What are the features of proteasomes?
- Only example of threonine protease family
- Major site of intracellular protein degradation
- Ubiquitin dependent
What are the pathways used to degrade proteins?
- Lysosomal pathway
- Ubiquitin-mediated
Where are the end products of lysosomal degradation exported to?
The cytosol
What are the 4 ways of delivery of substrate proteins to the lysosome?
- Endocytosis
- Autophagy
- Phagocytosis
- Chaperone-mediated autophagy
How does autophagy delivery substrate proteins to lysosome?
Macromolecules and/or organelles are engulfed by ER-dervied membrane and delivered to the lysosome
What do acid hydrolases require to ensure they are transported to the lysosome?
Mannose-6-phosphate tag added at the golgi
How are mannose-6-phosphate tags added to an N-linked oligosaccharide?
- Addition of GlcNAc to an N-linked oligosaccharide in cis-golgi
- Phosphodiesterase removes GlcNAc, leaving just the M6P tag
Why are incorrectly folded amino acids not tagged with Mannose-6-phosphate?
If not properly folded then the amino acids are not in the correct place for the signal patch so it won’t bind
What recognises the M6P tag?
Receptor proteins in trans-golgi network
How is the M6P tag recognised to allow acid hydrolase to be transported to lysosome?
- M6P tagged acid hydrolase binds to M6P receptor in TGN
- Clathrin coasted vesicle is transported to early/late endosome and then to the lysosome
- Low pH causes M6P tagged acid hydrolase ro dissociate from the receptor
How is the M6P recycled?
What can lysosomal storage diseases result in?
The formation of inclusion bodies
What causes the formation of inclusion bodies in lysosomal storage diseases?
- Problems with GlcNAc phosphotransferase
- Substrates gets delivered but enzymes do not so substrates are not degraded
What is the structure of ubiquitin?
- small 76 residue protein
- Lysine residues exposed on surface that are critical to function
How does ubiquitin attach to proteins?
Via isopeptide bond between ubituitin’s C-terminal carboxylate and lysine residue on substrate
What reads ubiquitin signals?
Ubiquitin binding domains on proteins
Why is the ubiquitin-lysine bond not a peptide bond?
It is not in the backbone of the protein
What must happen to ubiquitin before it can bind to substrate?
It must be activated
What kind of ubiquitination targets proteins to the proteasome for destruction?
Polyubiquitination
Which enzymes activate ubiquitin?
E1, E2, E3
What does E1 do to activate ubiquitin?
- C-terminal carboxylate of ubiquitin forms a thioester bond with a key cyteine residue of E1
- E1 then binds E2 which uses ATP
What does E2 do to activate ubiquitin?
- Ubiquitin conjugating enzyme
- The E1 to E2 shuttle moves the activated ubiquitin onto a key cysteine residue on E2
What does E3 do to activate ubiquitin?
- E3 is bound to E2
- E2/E3 complex binds to substrate protein and catalyses isopeptide bond formation
What is monoubiquitination used for?
Histone regulation
What is multiubiquitination used for?
Endocytosis
What are defects in E3 linked to?
Early onset Parkinson’s and breast and ovarian cancer (BRCA1 codes for an E3)
How are E3 enzymes activated?
- Requires ATP for activation by phosphorylation
- or allosteric activation by a ligand
How is a protein activated for ubiquitination?
- Activated via phosphorylation by a protein kinase
- or by unmasking a recognition site on substrate
- or removal of N-terminal residues to create a new destabilising N-terminal residue
What is the structure of the proteosome?
- Central catalytic core component which is hollow in middle
- Two caps
- Active sites inside central cavity of catalytic core
How is the catalytic core of the proteosome activated?
- Unfolded proteins can pass through the centre and make contact with the active sites located on the inside of the 20s core
What is the structure of the 20s catalytic core of the proteosome?
- composed of 2 x 7 membered rings of alpha subunits and 2 x 7 membered rings of beta subunits
Which beta subunits of the catalytic core of the proteasome are active?
B1, B2, and B5
What is unique about the proteasome protease mechanism?
Depends on nucleophilic attack by the N-temrinal threonine
How does the proteasome process proteins?
- 19s caps recognise ubiquinated protein and unfold it and feed it into central cavity using ATP (from ATP binding domains)
- 19s caps release the ubiquitin for reuse
What does the proteasome do to polyubiquinated proteins?
Hydrolyses them into small peptides
What happens to peptides leaving the proteasome?
- They are reused, and digested by cytosolic peptidases to amino acids for use in protein synthesis
- deaminated and used for different functions
Why is the unique protease mechanism in the proteasome useful?
Makes the beta units a good target for cancer as the proteasome can help maintain cell proliferation
