Intracellular Proteolysis

Question 1

What is a proteasome?

Answer 1

Proteasomes are very large protein complexes inside all eukaryotes and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm.
The main function of the proteasome is to degrade unneeded/damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases.
In structure, the proteasome is a cylindrical complex containing a ‘core’ of four stacked rings around a central pore.

Question 2

What are the four types of proteases?

Answer 2

• Serine proteases
• Cysteine proteases
• Aspartyl proteases
• Metalloproteases

Question 3

How do we classify proteases based on the manner in which they bind to and cleave their targets?

Answer 3

• ENDOPEPTIDASES: they break peptide bonds of non-terminal amino acids (ie. within the molecule)
• EXOPEPTIDASES: they break peptide bonds of terminal amino acids (ie. cleaves at the end of a polypeptide chain)

Question 4

What are the two subdivisions of Exopeptidases?

Answer 4

• AMINOPEPTIDASES: cleaves peptide bonds at the N-terminal (amine terminus)
• CARBOXYPEPTIDASES: cleave peptide bonds at the C-terminal (carboxy terminus)

Question 5

How specific can sequence specificity of proteolysis be?

Answer 5

The sequence specificity of proteolysis can be specific (protein activation, eg. insulin) or non-specific (protein degradation, eg. lysosomes).

Question 6

Give an example of protein activation by proteolysis.

Answer 6

(these have to do with serine proteases)
An example of protein activation by proteolysis would be the clotting factors.
The blood clotting system (coagulation pathway) is a proteolytic cascade. Each enzyme of the pathway is present in the plasma as a zymogen (inactive form), which, on activation, undergoes proteolytic cleavage to release the active factor from the precursor molecule.
The whole point of this pathway is to make Factor X, which catalyses the conversion of Prothrombin to Thrombin, which then continues on to make the clot.

Question 7

What is the cause of X-linked haemophilia?

Answer 7

Deficiencies in Factor VIII or IX of the coagulation pathway are the cause of X-linked haemophilia.

Question 8

What are some examples of Cysteine proteases?

Answer 8

• Bromelain
• Papain

Both can be used a meat tenderisers.

Question 9

What is an example of a known Aspartyl protease?

Answer 9

• HIV-1 Protease (retropepsin)

HIV-1 Protease cleaves poly-protein precursors to form functional proteins.

Question 10

What is ubiquitylation?

Answer 10

It is the addition of the ubiquitin ‘tag’ that targets the enzyme to the proteasome for degradation.

Question 11

What are the three enzymes we need to know involved in ubiquitylation?

Answer 11

E1: Ubiquitin-activating Enzyme
E2: Ubiquitin-conjugating Enzyme
E3: Ubiquitin-protein Ligase

Question 12

Describe the three-step process of ubiquitylation.

Answer 12

1) ACTIVATION: formation of a thioester bond between the COOH terminus of ubiquitin and a cysteine in E1 (requires ATP)
2) CONJUGATION: transfer of ubiquitin from cysteine onto E2
3) LIGATION: transfer of ubiquitin from E2 to a lysine in the target protein, or to the other ubiquitin molecules already attached to a target (polyubiquitination)

Question 13

What is the N-end rule, and what does it state?

Answer 13

The N-end rule is a rule that governs the rate of protein degradation through recognition of the N-terminal residue of proteins. The rule states that the N-terminal amino acid of a protein determines its half-life.