Intracellular Proteolysis

Question 1

What are the 4 classes of proteases?

Answer 1

• Serine proteases
• Cysteine proteases
• Aspartyl proteases
• Metalloproteases

Question 2

Describe two ways of classifying proteases

Answer 2

Endopeptidases v exopeptidases

Specific v non specific recognition

Question 3

Describe the difference between endopeptidases and exopeptidases

Answer 3

1. Endopeptidases cleave protein in the middle somewhere in the chain Exo cleaves the protein right at the end- amino terminus. and carboxyl terminus- aminopeptidases, and carboxypeptidases

Question 4

Describe the difference between proteases that recognise specific and non-specific amino acid

Answer 4

Recognize specific amino acid in the substrate. The hydrolysis activates the protein.

If nonspecific- protein degradation- food stuff digestion in the small intestines or lysosomes by organelle called proteasome

Question 5

What are the inactive proteins?

Answer 5

Zymogen
Proprotein
Proenzyme

Question 6

Give an example of protein activation by proteolysis in the stomach(4)

Answer 6

trypsin cleaves amino acids from chymotrypsinogen and it becomes active chymotrypsin, and three amino acids are removed so three separate peptide chains are made which are joined by disulfide bonds–> alpha chymotrypsin .

Trypsin which is involved in the activation of chymotrypsinogen must itself be activated by enteropeptidase

Question 7

Decsribe an example of protein activation by proteolysis in the Golgi(2)

Answer 7

Proopiomelanocortin in the Golgi which is a pro hormone and is produced in the Er and processed in the GA through proteolysis.

Depending on the cell type a very large numbers of different peptide hormones can be generated from one sequence of proteins.

Question 8

How is proteolysis involved in the production of insulin?(3)

Answer 8

1. Proteolysis-
   Removal of signal sequence in the signal peptide in thepreproinsulin- turned into proinsulin
   Proinsuliniscleavedinto three peptides- A, B, C chain.
   Diff chains have diff amino acids

2.Disulfide
Removal of C chain
Disulphide bridges between A and B chain
Comprising 51 amino acids.

3.Glycosylation anddeyglycosylation
Addition of sugars to the protein or removal
Also finalised in the Golgi apparatus
leaves recognition signals for lysosome proteins
Determination of destination of the proteins occurs here
Phosphorylation indeglycosylationis for secretory proteins

Question 9

How is protein activation involved in clotting factors?(4)

Answer 9

1. Kallikren cascade
2. The activation of factor 12 is triggered by damages surfaces,
3. after proteolysis of factor 12 it becomes active and goes on to activate other enzymes.
4. Prothrombin is cleaved into thrombin and is made active by factor 10.

Question 10

Describe how the royal disease arise(5)

Answer 10

·Introns require specific AG splice acceptor site, so it creates AG where there used to be an AA, so it creates a new spliceosome acceptor site.

· There is a shift in the reading frame so all the amino acids are completely different so there is a nonfunctional factor 9.

· Important when there is damage cell leading fibrin clot

· Involves series of cascade all with enzymes that are serine proteases.

· At each step one serine protease cleaves to another serine protease converting inactive zymogen to the active protease

Question 11

What are some examples of cysteine proteases?

Answer 11

Bromelain, Papain- seasoned meat tenderizer.

Partially break down the fibers making it easy to chew.

Question 12

How is HIV-1 protease involved in protein activation?

Answer 12

The HIV genome generates two proteins Gag and Pol they need to be activated by proteolysis because they are both proproteins

2. The enzyme that catalysises HIV-1 protease cleaves Gag and Pol- if this enzyme were not available it would be inactive

Question 13

What is the drug for HIV?

Answer 13

HAART = “highly active antiretroviral therapy”Discovery of Gag and Pol lead to its purification and crystallizing and drugs/ inhibitors that prevents this cleavage.

Question 14

What are the two ways of protein degradation?

Answer 14

Lysosomal degradation is one pathway which requires acidic lysosome enzymes,

the other pathway is the ubiquitin proteasome pathway.

Question 15

Why is protein degradation compartmentalised?

Answer 15

Nonspecific protein degradation is tightly compartmentalized, so enzymes do not cause damage if it was not tight.

2. Compartmentalized in lysosomes or the proteasome via ubiquitylation Proteases designed in lysosome are designed to minimize damage through low pH so would not work at neutral conditions

Question 16

What must happen to protein needing degradation by the proteosome?

Answer 16

Covalently modified with ubiquitin molecules chain and is then targeted to the proteasome where degradation occurs.
The ubiquitin targets the inside of the proteasome.

Question 17

Describe ubiquitin(2)

Answer 17

Ubiquitin is highly conserved small protein 76 amino acids and weight on 8.6 kDa.

2. small regulatory proteins that are attached covalently to the substrate protein.

Question 18

Describe the ubiquitination of protein marked for the proteasomes(6)

Answer 18

Ubiquitin-activating enzyme (E1)

1. Ubiquitin attaches to enzyme called E1(Ubiquitin activating enzyme)
2. E1 reacts with the carboxy terminal via the thiol group on the cystine residue – requires ATP.
3. Generates a thiol-ester bond Ubiquitin-conjugating enzyme (E2)
4. Transfer of ubiquitin to a cysteine on E2(ubiquitin conjugation enzyme) via the thiol bond Ubiquitin-protein ligase (E3)
5. · Transfer of ubiquitin from E2 to a lysine on the target protein

6· Many different E3s exist that recognize specific target proteins

Question 19

What marks proteins for degradation in the proteosome?

Answer 19

Polyubiquitylation via K11 or K48 marks proteins for proteasomal degradation.

ubiquitin molecules are attached to lysine 48 or 11 on the ubiquitin chain.

Question 20

What happens to the ubiquitin after the protein is degraded?(2)

Answer 20

A large protease complex called the proteasome orthe 26S proteasome digests the ubiquitinated proteins .

The proteasome is a very large protease complex, the active sites points towards the inner cavity. The ubiquitin targets the inside of the proteasome.

Question 21

How many ubiquitin molecules have to be attached to the protein to be marked for the proteosome

Answer 21

At least 4

Question 22

What are the products of the degradation?(3)

Answer 22

Ubiquitinated proteins are processed to peptide fragments from which the ubiquitin is subsequently removed and recycled.

The peptide fragments are further digested to yield free amino acids, which can be used for protein synthesis.

the amino group can be removed and processed to urea and the carbon skeleton can be used to synthesize carbohydrate or fats or used directly as a fuel for cellular respiration.

Question 23

Why are there varying half lives?

Answer 23

Varying half-lives is to do with the very first amino acids at the end of the N terminus

The first amino acid is methionine but is quickly (sometimes) removed exposing the second amino acid. The structure of the second has a great influence on the stability of the protein.

These amino acids are recognized by ubiquitin ligases

Question 24

What are the enzymes involved in degradation of the first amino acid?

Answer 24

U3 ubiquitin ligases

Question 25

What is a zymogen?

Answer 25

A proenzyme that is a precursor of an enzyme

Question 26

Recall some stabilising and destabilising amino-terminal amino acid residue

Answer 26

Stabilising- met, gly, ser

Destabilising- Tyr, glu, pro

Question 27

what does formation of the isopeptide bond require?

Answer 27

ATP