Proteases 5

Question 1

What are the different approaches in which protease inhibition can be obtained?

Answer 1

Canonical inhibitors where the compound binds to the enzyme as if it was a substrate and blocks substrate cleavage
Exocite binding inhibitors inhibitor binds close to but not at the site of substrate binding blocking it
Quasi-Substrate inhibitors which is a combination of exocite binding and canonical inhibitors
Allosteric inhibitors where the inhibitor binds away from the active site preventing dimerization or activation of the protease

Question 2

What are the endogenous inhibitors of proteases?

Answer 2

Serpins which inhibit serine proteases
Cystatins, Calpains IAP family inhibits cysteine proteases
Renin Binding protein which inhibits aspartic protease renin
Tissue Inhibitor of Metalloproteases which inhibit metalloproteases

Question 3

How can protease activity be modulated?

Answer 3

pH change, redox state, alkylation and metal ions

Question 4

What are the two key serpins?

Answer 4

Serpin A1 (Alpha-1 antitrypsin) which is produced in the liver, transported to the lungs
It inhibits neutrophil elastase which modulates inflammation protecting the lungs to prevent damage to the tissue
Serpin c1 (antithrombin) controls blood clotting through inhibition of thrombin and other coagulation proteins

Question 5

How does blood clotting occur and how does protease inhibition influence this process?

Answer 5

When damaged the inner layer of the blood vessel releases tissue factor which complexes with coagulation factor VIIa and activates factors IXa and Xa which leads to activation of serine protease pro-thrombin which is cleaved to active thrombin leading to fibrinogen conversion to fibrin to form an insoluble meshwork
Serpin C1 inactivates thrombin through trapping it in a 1:1 ratio of thrombin:antithrombin
When bound antithrombin makes the active site inaccessible to its usual substrate. Herapin can increase the rate of this inhibitors activity

Question 6

Why are there many more proteases than protease inhibitors?

Answer 6

Not all proteases have an endogenous inhibitor and some inhibitors can affect more than one protease

Question 7

What are calpains and how are they inhibited?

Answer 7

non-lysosomal calcium-activated, neutral proteases found in various tissues in mammals and other organisms, associated with loads of functions
Inhibited by calpastatin forming the calpain system

Question 8

What are cystatins?

Answer 8

proteins that contain multiple cystatin-like sequences (but not all are active cysteine protease inhibitors)
Includes the stefin family, cystatin family, kininogens
Potent inhibitors of cysteine proteases of the papain family and form equimolar, tight, reversible complexes with human cysteine proteases

Question 9

What are the functions of protease inhibitors in the stefin family?

Answer 9

Cystatin A inhibits cathepsin L and cathepsin B, and is in effect a serum biomarker for cancer; Cystatin B inhibits cathepsin L, cathepsin B and cathepsin H, and is thought to protect the cells from leakage of cathepsins from the lysosomes

Question 10

What are the functions of protease inhibitors in the cystatin family?

Answer 10

ubiquitous Cystatin C produced in most cell types and organs, secreted to cerebrospinal fluid and plasma, is a biomarker for a range of conditions.Cystatin C inhibits cathepsins L, H, B and S, as well as other proteases

Question 11

What are the endogenous aspartic protease inhibitors?

Answer 11

Pepsin does not have an endogenous inhibitor but activation is regulated by pH
Cathepsin D has no known endogenous inhibitor in mammals
Renin of the Renin-Angiotensin System (RAS) is inhibited by Renin binding protein (RnBP) inhibits renin activity by forming a dimer with renin, known as high molecular weight renin this complex is unlikely to have a key role in modulating RAS in vivo due to the cytoplasmic location of RnBP

Question 12

What are the endogenous metalloprotease inhibitors?

Answer 12

Tissue Inhibitor of MetalloProteinases (TIMPs) complexes and irreversibly inactivates MMPs by binding to their catalytic zinc cofactor.
These inhibitors have considerable overlap in their targets

Question 13

How is proteasome activity regulated?

Answer 13

Proteasome activity is regulated by a number factors, including availability of ubiquitinated substrate, O-GlcNAc modification and ATP levels not an endogenous molecule

Question 14

What is the role of pharmacological protease inhibitors in anti-HIV drugs?

Answer 14

A number of drugs inhibiting the HIV protease have been developed and approved for clinical use. Successful inhibition of this protease stops production of new virus particles since the new polyprotein chains need to be cleaved in order to liberate the proteins needed for the next generation of viruses.

Question 15

Why is it important to include protease inhibitors in biological experiments?

Answer 15

Proteases are active but regulated and compartmentalized in living cells. Once a biological sample is removed from its context, these preventive measures are disrupted and endogenous proteases will start destroying the sample. This is a major hindrance to the study of proteins from biological samples

Question 16

What aspects are important to consider when determining which protease inhibitors to use for lab experiments?

Answer 16

If you are studying a specific protease or a require a protease to act as part of the experimental method then a generic cocktail which will inhibit all proteases will not be useful
Working with samples at low temperatures can also be useful as it will reduce protease activities

Question 17

What are the common components of cocktails of protease inhibitors that can be used for lab experiments?

Answer 17

Aprotinin which is an inhibitor of serine proteases
Bestatin which is a selective inhibitor of various amino peptidases
E-64 which is a potent irreversible inhibitor of cysteine proteases
Leupeptin which inhibits serine and cysteine residues
Pepstatin A is an inhibitor of acid proteases