intracellular proteolysis

Question 1

what is proteolysis

Answer 1

• Proteolysis is the reversal of protein synthesis in a condensation reaction.
• Peptide bonds are cleaved with the consumption of water.

Question 2

what are the two classes of proteases ?

Answer 2

• Endopeptidases – cleave proteins in the middle of the polypeptide chain
• Exopeptidases – remove one or two amino acids from the end of the polypeptide chain. If they work in the amino end, they are called aminopeptidases, and exopeptidases that work on the other end of the polypeptide chain are called carboxypeptidases.

Question 3

what will happen If proteases recognise a specific amino acid sequence in the substrate

Answer 3

the cleavage will result in protein activation generally.

Question 4

what will happen if proteases are non-specific ?

Answer 4

protein degredation

Question 5

give an example of protein activation by proteolysis via digestive enzymes

Answer 5

• the activation of chymotrypsin
  • Chymotrypsinogen is initially an inactive protein with 245 amino acids, and is cleaved and recognised by the protease trypsin, which cleaves between amino acids 15 and 16.
• This generates pie-chymotrypsin. It is active and can cleave itself at another site at 146 and 148. This generates 3 peptides that are connected by disulfide bonds to produce the final active alpha chymotrypsin.
• Trypsin itself must be activated through proteolysis. This involves enteropeptidase which yields the active trypsin.

Question 6

explain the Proteolytic processing of proopiomelanocortin in the Golgi

Answer 6

-gives rise to multiple peptide hormones
• Proopiomelanocortin is a prohormone translated in the ER and then processed in the golgi through proteolysis.
• Generating many peptide hormones from the single precursor protein.

Question 7

explain the protein activation by proteolysis of insulin

Answer 7

• Insulin must also be processed by proteolysis before it can be active
• The initial protein that synthesises at the er is preproinuslin which carries a singal sequence that directs it to the RER during synthesis
• The removal of the signal sequence produces a proinsulin which acquires disulphide bonds.
• This must be cleaved again to release the internal fragment called the c peptide and produce the final mature insulin.

Question 8

explain the protein activation by proteolysis of clotting factors

Answer 8

• The clotting cascade involves serine proteases.
• At each step of the cascade one serine protease cleaves another serine protease, converting an inactive zymogen to the active protease.
• Factor XIII is a transglutaminase

Question 9

which 2 factors are linked to x-linked haemophillia

Answer 9

• 2 factors in the clotting cascade, factors 9 and 8 are X linked genes, and mutations will impair clotting and lead to x-linked haemophilia.

Question 10

how does x-linked haemophilia arise ?

Answer 10

• There were no mutations in the coding region, but there was a single point mutation in the introns, a to g, close to the splicing acceptor site. This creates a new splice acceptor site ( as it requires usually ag at the 3’ end) which then cause the frameshift of 2 nucleotides , changing the amino acid sequence and the introduction of a stop codon.

Question 11

give 2 examples of famous cysteine protease

Answer 11

• Bromelain, papain.

Question 12

How does HIV-1 protease work ?

Answer 12

• HIV-1 protease (retropepsin)
• HIV-1 VIRUS From its genome expresses large precursor proteins, Gag and Pol that must be cleaved, processed and activated proteolytically in order to produce a number of protein products.
• The enzyme that catalyses the proteolytic activation process is the hiv-1 protease, an aspartly protease.
• If the enzyme is unavailable then the virus is inactive
• Discovery of this lead to its purification and determination of its crystal structure. Based on its crystal structure drugs were developed

Question 13

protein degredation is tightly ? and why?

Answer 13

• Protein degradation is tightly compartmentalized.
• If degradative proteases were able to diffuse freely around the cytoplasm, they would cause a lot of damage to proteins that aren’t meant to be digested.

Question 14

where are proteases found ?

Answer 14

• Proteases that have low specificity ( can degrade any protein substrate ) are found in the GI tract and inside cells are compartmentalized either in lysosomes or inside a barrel shaped structure called the proteosome.
• Proteases formed in lysosomes operate in a low pH to minimise potential damage. If they were to escape from the lysosome into the cytoplasm, they would not be active as the pH is close to neutral in the cytoplasm.

Question 15

what is the ubiquitin proteosome pathway

Answer 15

where a protein that needs to be degraded and is found in the cytoplasm or nucleus is first covalently modified with a chain of ubiquitin molecules, and is then targeted to the proteosome, where degradation takes place.
• The products formed : amino acids + small peptides.

Question 16

what is a ubiquitin

Answer 16

• A small protein
• Highly conserved in evolution, similar eg between yeast and humans.
• It has 76 amino acids and a molecular weight of 8.6 kDa
• It’s the attachment of ubiquitin to the target protein that marks them for degradation in the proteosome.
• This attachment occurs in 3 steps, involving 3 types of enzymes.

Question 17

outline the steps in protein ubiquitylation

Answer 17

1) Ubiquitin is attached to an enzyme called E1, ubiquitin activating enzyme, which reacts with the carboxy terminal via the thiol group on the cystine residue on the E1. This reaction requires ATP and generates a thioester bond.
2) The ubiquitin is transferred from E1 to E2, ubiquitin-conjugating enzyme. Ie its being transferred from one cysteine to another and is still attached via a thioester bond.
3) Through the aid of the E3 enzyme, ubiquitin ligase, the ubiquitin is transferred onto the lysine residue of the target protein.
4) It is the ubiquitin ligases that confer the specificity in this process. They recognise only specific substrates and are often regulated, such that they ubiquitinate and therefore mark their proteins for degradation.

Question 18

what marks proteins for proteasomal degredation ?

Answer 18

Polyubiquitylation via K11 or K48 marks proteins

Question 19

explain what polubiquitylation is

Answer 19

• If a protein is to be degraded through ubiquitination, a chain must be formed where additional ubiquitin molecules are attached to either lycine 48 or lyicine 11 on the ubiquitin chain. A minimum of 4 ubiquitin molecules must be attached in order for the protein substrate to be marked for proteolysis.

Question 20

what are the Components of the ubiquitinating enzyme complex.

Answer 20

Ubiquitin-activating enzyme (E1)
•Formation of thioester bond between COOH terminus of ubiquitin and a cystein in E1
•Reaction requires ATP

Ubiquitin-conjugating enzyme (E2)
•Transfer of ubiquitin to a cystein on E2

Ubiquitin-protein ligase (E3)
•Transfer of ubiquitin from E2 to a lysine on the target protein
•Many different E3s exist that recognize specific target proteins

Question 21

Why do some proteins have short lives and some have long lives. How is this possible ?

Answer 21

The N-end rule

Question 22

explain the N-end Rule •

Answer 22

• This often has to do with the first amino acid at the n terminus of the proteins.
• The first amino acid in a protein is always methionine. The first methionine is often very quickly removed, exposing the second amino acid in the polypeptide chain. It is the nature of this amino acid that has a great influence on the stability of the resulting protein. This is because the amino acids are recognised by E3 Ubiquitin ligases.
• Some amino acids are very stabilising, e.g met, gly, ala, ser, thr, val. This is because they are not very well recognised by the E3 ubiquitin ligase. If the E3 does not recognise those amino acids, the proteins will not be ubiquitinated and hence will not be degraded
• Other amino acids which are destabilizing, - they are effectively recognised by E3 ubiquitin ligases. The ligase has a great affinity for these amino acids, and then will be more frequently ubiquitylated and hence marked for degradation.
• N-end rule U3 ubiquitin ligases have poor affinity for stabilizing amino-terminal residues.

Question 23

what are cathepsins?

Answer 23

lysosomal proteins

Question 24

why do proteases in the small intestine need to be non specific?

Answer 24

so they can degrade any type of protein that is ingested

Question 25

what is the activation of factor 12 in the clotting cascade activated by?

Answer 25

damaged surfaces

Question 26

What are the two pathways for protein degradation?

Answer 26

Lysosomal degradation and the other is the ubiquitin proteasome pathway

Question 27

What is the proteasome?

Answer 27

a large protease complex the active sites all point towards the center of the inner cavity.

Question 28

How many ubiquitin molecules need to be added to the bound ubiquitin

Answer 28

4

Question 29

what determines how stable a protein is ?

Answer 29

the affinity of the ubiquitin ligase (Km) to the amino acid that comes after methionine

Question 30

If the affinity of the ubiquitin ligase to the amino acid after methionine is high what does this mean

Answer 30

that the protein has a higher chance of being ubiquitylated and being degraded.