Lecture 16 - Intracellular proteolysis Flashcards
Proteolysis (1)
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Proteolysis is typically catalysed by cellular enzymes called proteases.
Proteases = Proteinases = Peptidases = Cathepsins (7)
Serine proteases
Cysteine proteases
Aspartyl proteases
Residues of 2/3 play a part in catalysis process at the active site of an enzyme.
Metalloproteases - complexes with metal ions.
Endopeptidases- Enzyme cleaves substrate protein in middle of peptide chain/ breaks peptide bond.
Exopeptidases – cleave 2/3 amino acids from end of amino acids.
Protein activation by proteolysis (5)
SPECIFIC - enzyme recognises a certain sequence of amino acids and will only cleave if this is present.
NON-SPECIFIC - Protein degradation e.g. small intestine of protein food (steak/eggs).
Once inactive enzymes undergo cleavage by proteases it becomes an active protein.
(Inactive) Zymogen/Proprotein/Proenzyme –> (Active) Enzyme/Protein.
Prothrombin –> Thrombin.
Protein activation by proteolysis - Digestive enzymes (5)
Chymotrypsinogen and trypsinogen are synthesised on the ribosomes of the RER.
π -chymotrypsin and trypsin are involved in protein degradation and digestion.
Chymotrypsinogen (Precursor/Inactive) activated by trypsin into π -chymotrypsin.
π -chymotrypsin undergoes autocatalysis and cleave it self producing 3 chains joined by disulphide bonds.
Protein activation by proteolysis - Clotting factors (3)
- Amplifying cascade.
- Activation of factor 12, activates factor 11 and so on.
- Factor 10 helps get prothrombin to thrombin.
Haemophilia B (6)
They found a single-nucleotide change in the gene for clotting factor IX that causes incorrect RNA SPLICING and produces a truncated, non-functional protein.
Insertion of 3 base pairs (codon) [mutation of A to G and insertion of AG] this causes a change in the reading frame. Codes for a different sequence of amino acids.
Queen Victoria had haemophilia.
Deficiencies of Factor VIII or IX are the Cause of X-Linked Haemophilia. Disease gene was found on the X chromosome, so her daughters were carriers, who passed this down to their children.
Factor IX (9) and is part of the clotting cascade.
Christmas disease, named after Stephen Christmas, the first patient described with this disease.
Protein proteases - Cysteine (2)
Bromelain, Papain – seasoned meat tenderiser, a mixture of proteases (cystine).
Will digest some of the collagen and some of the extracellular matrix and make food easier to chew.
Protein proteases - Aspartyl (4)
HIV-1 protease (retropepsin).
Gag and Gag-Pol are proenzymes that need to be processed by proteolysis in order to be activated.
HIV protease cleaves newly synthesised polyproteins (Gag and Gag-Pol) at nine cleavage sites to create the mature protein components of an HIV virion.
HIV virion is the infectious form of a virus outside of the host cell. Without effective HIV protease, HIV virions remain uninfectious.
Proteasome (2)
Proteasome (26S) – Large protein machine that degrades proteins that are damaged, misfolded, or no longer needed by the cell.
Its target proteins are marked for destruction primarily by the attachment of a short chain of ubiquitin.
Protein degradation (8)
Chain of ubiquitin attached covalently to a substrate.
Phase 1: Protein adapted by ubiquitylation.
Phase 2: Degradation.
Lysosomal degradation is usually unspecific.
Ubiquitylation proteosome pathway: PHASE 1: Ubiquitylation E1: Ubiquitin-activating enzyme E2: Ubiquitin-conjugating enzyme E3: Ubiquitin ligase
Ubiquitylation steps (4)
1) Thioester bond forms between ubiquitin (COOH/carboxyl) and a cysteine E1 (HS). Using ATP.
2) Ubiquitin transfer to cysteine using E2.
3) Ubiquitin on E2 transferred to target protein by E3.
4) Different E3 enzymes exist for specific target proteins.
Half-lives of selected proteins (hours) (4)
Short lived
Ornithine decarboxylase = 0.2
RNA Polymerase I = 1.3.
Long-lived
Aldolase = 118.
Cytochrome C = 150.
Half-life (1)
Time it takes to degrade half the protein that was present at the beginning.
N-end rule () Half-lives (4)
Explains why some proteins are long/short-lived.
Methionine is the start codon, usually cleaved off by methionine aminopeptidases.
First AA after this determines half-life/stability of protein.
As protein is recognised by E3 (ubiquitin ligase) the affinity for the n-terminus determines its turnover.
High affinity = High ubiquitylation and degradation = Destabilising amino-terminal residues.
e.g. Tyrosine - 10 minutes. Arginine - 2 minutes.
Low affinity = Low ubiquitylation and degradation = Stabilising amino-terminal residues.
e.g. Alanine/Glycine/Methionine - 20 hours.
Statins block Cholesterol synthesis through inhibition of HMG-CoA Reductase (3)
Atorvastatin (Lipitor) Fluvastatin (Lescol). Lovastatin (Mevacor, Altocor, Altoprev). Pitavastatin (Livalo, Pitava). Pravastatin (Pravachol). Rosuvastatin (Crestor). Simvastatin (Zocor).
