Biology subjects

Bolgi, O.

Publications and source records attributed to Bolgi, O..

3 recordsLinked to original sources

bioRxiv2025

Specificity profiling of SARS-CoV-2 PLpro using proteome-derived libraries of linear peptides suggests secondary preference for basic motifs

SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and immune modulation. Here, a proteomic identification of protease cleavage sites (PICS) approach was applied using proteome-derived peptide libraries to determine the enzymes substrate specificity. PLpro exhibited a yet unreported preference for basic amino acids at P1, primarily arginine or lysine. This secondary specificity frequently involved cleavage between two basic residues (e.g., K|K, K|R or R|K). Experiments with in-house and commercial PLpro in GluC-generated peptide libraries from Escherichia coli and HEK293 proteomes confirmed this preference, though with lower overall efficiency compared to typical trypsin-like proteases. SARS-CoV-1 PLpro likewise displayed this basic-site specificity, underscoring its conservation across related coronaviruses. Site-directed mutagenesis of acidic residues near the catalytic triad to neutral variants altered cleavage preferences, indicating the involvement of these side chains in substrate binding and potential alternative binding modes. We also evaluated wild-type PLpro specificity on intact protein lysates rather than peptide libraries to assess how structure influences cleavage patterns. Notably, P1 arginine specificity persisted at the protein level, whereas lysine specificity was reduced, suggesting additional structural constraints in complex substrates. A strong presence of glycine on the prime side further suggests a bias toward unstructured regions. These findings reveal an expanded substrate recognition repertoire for SARS-CoV-2 PLpro, which may be relevant for the design of targeted inhibitors and understanding of viral protease function.

biochemistry↗

bioRxiv2020

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

microbiology↗

bioRxiv2020

Dipeptidyl peptidase 9 triggers BRCA2 degradation by the N-degron pathway to promote DNA-damage repair

Dipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development.

biochemistry↗