Intracellular proteolysis Flashcards
What is intracellular proteolysis?
The breakdown of proteins or peptides into amino acids by the action of enzymes
Describe the classes of proteases
- Proteases break down of protein (with proteases: cleave the peptide bond).
- Aspartic /Cystine -the residues play an important role in catalysis process at the active site of the enzymes.
- Metalloproteases – Form 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. These break down the ends of the protein
Describe protein activation by proteolysis
- SPECIFIC- enzyme recognizes only specific a certain sequence of amino acids in its substrate. Only cleave the substrate if a certain sequence required is preset for it to bind to.
- NON-SPECIFIC- Protein degradation e.g. small intestine degradation of protein foods (e.g. steak/eggs).
- Inactive enzymes once it undergoes cleavage by signal protease it become an active protein. e.g. Prothrombin to thrombin -> proprotein to active protein, this is an example of proteolysis.
Describe protein activation of proteolysis in digestive enzymes
- Chymotrypsinogen and trypsinogen are synthesised on the ribosomes on the RER, they are secreted enzymes which need to travel to plasma membrane.
- π-chymotrypsin and trypsin are involved in protein degradation in digestion.
- Precursor of π-chymotrypsin is Chymotrypsinogen initially a peptide of 245 amino acids. Then trypsin activates it cutting itat the 15th amino acid (Arg) separating it from the 16th (Ile- Leucine ). Then π-chymotrypsin undergoes autocatalysis and cleaves itself removing … [as shown in image]. Producing 3 chains connected by disulphide bonds.
Describe protein activation of proteolysis in blood clotting
- Amplifying cascade.
- Activation of factor 12, activates factor 11 and so on.
- Factor 10 helps get prothrombin to thrombin
Describe Hemophilia
- Factor IX (9) and is part of the clotting cascade.
- Queen Victoria had haemophilia. She passed down this disease to her family. 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.
- This disease is called haemophilia B. 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.
- Sometimes called Christmas disease, named after Stephen Christmas, the first patient described with this disease.
Describe prominent proteases (clue = Bromelain and Papain.)
Prominent proteases
Cysteine
• 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.
Aspartyl
• HIV-1 protease (retropepsin) – Gag and Gag-Pol are pro proteins 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, the infectious form of a virus outside of the host cell. Without effective HIV protease, HIV virions remain uninfectious.
• Number of people with HIV increases steadily until 1995, into of HIV inhibitors there is a huge decrease in number of people with disease.
Describe protein degradation
- 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.
- Chain of ubiquitin attached covalently to a substrate.
- Phase 1: Protein adapted by ubiquitylation
- Phase 2: degradation
- Lysosomal degradation is usually unspecific
Describe the ubiquitylation proteasome pathway
• PHASE 1: Ubiquitylation
• E1: Ubiquitin-activating enzyme
• E2: Ubiquitin-conjugating enzyme
• E3: Ubiquitin ligase
1. Forms thioester bond between end (COOH) group of ubiquitin and a cystein E1 (HS). Using ATP.
2. Transfer of ubiquitin to a cystein on to E2.
3. E3 transfer ubiquitin from E2 to target protein.
4. Many different E3 enzymes exist for specific target proteins.
Describe the N-end rule
- Explains why some proteins are short/long lived.
- Methionine is the start codon it is usually cleaved off by methionine aminopeptidases.
- The first amino acids after methionine determines a proteins half-life/stability. This is because the protein is recognised by ubiquitin ligase the affinity of ubiquitin ligase for the n-terminus of the protein that determines it turnover.
- High affinity = higher chance of ubiquitinalation and degraded
- N-end rule U3 ubiquitin ligases have poor affinity for stabilizing amino-terminal residues
Describe the SREBP cycle for cholesterol regulation
- There are three main proteins in the ER (yellow, green and purple).
- When the cell needs cholesterol, the easiest way is to get it from LRL from outside the cell. You have increased expression of the LDR gene in order to get more LDRs into the cell.
- SCAP measures the amount of cholesterol.
- SREBP moves the Golgi apparatus through vesicular transport.
- At the Golgi apparatus, 2 proteases are activated to cleave the SREBP protein. SREBP is a transcription factor protein, it will bind to a gene in the nucleus to activate the transcription of a gene, I t bind to the promotor of the LDR promotor gene. This leads to uptake of cholesterol and higher cholesterol levels are restored.
- In order to reduce it you have the blocking of the SREBO to the nucleus.
