Proteases regulate the activation and deactivation of SARS-CoV-2 peplomer

Proteases are enzymes that catalyze the breakdown of proteins by hydrolysis and are key to the infectious process of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).The· Spike protein Those that first interact with the ACE2 receptor on the surface of the host cell must undergo cleavage to initiate a conformational change that allows entry into the cell. Proteolysis has been shown to be accelerated and occur at several stages. For example, by a membrane-bound protease belonging to the host cell. TMPRSS2Extracellular proteases such as furin; or intraendosomes after endocytosis Cathepsin B and L, allowing the virus to escape to the cytoplasm.

The above proteases have been shown to be involved in the activation of the SARS-CoV-2 peplomer, but other proteases may not yet be identified.Research papers recently uploaded to the preprint server bioRxiv* Depends on Pratt et al. (17^th (February 2021) Cathepsins B, K, L, S, and V were highlighted as potential leads, with the aim of identifying these enzymes by computational screening and molecular docking simulations. Interestingly, some existing conditions associated with poor outcomes in COVID-19 patients are also associated with elevated levels of these cathepsins, such as diabetes and cardiovascular disease. Additional analysis by production of recombinant peplomer protein and incubation with the enzyme of interest demonstrated the validity of the computational model.

What method was used to identify the enzyme?

This group uses a program called Protease-Acid Cleavage from MEROPS ANalyzed Specificities (PACMANS) to identify potential cleavage sites by searching for specific amino acid sequences, SARS-CoV-2 peplomer. I started by analyzing. The program also scores and ranks cleavage sites based on their affinity with the enzyme of choice, allowing the group to perform additional docking simulations at these specific sites.

As mentioned above, furin and cathepsins B and L are already involved in the cleavage of peplomer proteins, and this process confirmed their involvement. Furin has been shown to have the most highly rated cleavage site of the enzymes tested and is located at the S1 / S2 subunit site, which causes the conformational changes required for membrane fusion. Other enzymes of interest with cleavage sites in the S1 / S2 region include cathepsins B and K, which primarily target the region of the S1 or S2 domain, or the seven repetitive domains of peplomer. did.

PACMANS only analyzes amino acid sequences without considering the three-dimensional structure of the protein. Therefore, the enzymes of interest (furin and cathepsin B, K, L, S, and V) were simulated in molecular docking programs for both open and closed conformational peplomer proteins. The proximity of the enzyme’s active site to cleavage site is assessed three-dimensionally, with cathepsins L and K closest at about 0.3 nm and farthest still at 1.1 nm, generally close enough to initiate binding. ..

Recombinant forms of full-length peplomer were generated and incubated with the enzyme for various times to visualize cleavage fragments. Each cathepsin generated its own cleavage fragment to support the calculation results. Interestingly, the enzymes already known to be involved in the cleavage of peplomers (cathepsin B and L) actually produce the fewest unique immunodetectable products, with the most cathepsin K and V. Generated.

What does that mean?

In this group, multiple simultaneous cleavage events in the N-terminal domain and the receptor binding domain have an inactivating effect and do not conform to the protein, beyond the activation cleavage events observed around the S1 / S2 site. It is assumed that it may stabilize and prevent cell invasion. Fragments released from the N-terminal domain after cleavage were identified here, confirming that their removal could lead to destabilization and unfolding of peplomer proteins.

This study highlights the various roles these proteases play in the activation, deactivation, cell invasion, and pathogenicity of the SARS-CoV-2 virus and provides deeper insight into the progression of COVID-19 disease. Presents a methodology that allows screening of other enzymes. ..

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