Biology subjects

Mar, E.

Publications and source records attributed to Mar, E..

3 recordsLinked to original sources

bioRxiv2026

Cathepsin K Mediates the Formation of Potential Rheumatoid Arthritis-Relevant Cis- and Trans-Spliced Peptides Compatible With HLA-DR4 Presentation

Rheumatoid arthritis (RA) is characterized by a loss of immunological tolerance to synovial self-proteins, yet the initial triggers generating novel neo-antigens remain incompletely defined. Here, we demonstrate that human cathepsin K (hCatK), a key cysteine protease driving joint degradation in RA, catalyzes covalent cis- and trans-splicing of peptides from major RA-associated self-proteins and foreign antigens, including type II collagen, fibrinogen, and SARS-CoV-2 Spike protein. Using high-resolution LC-MS/MS and database-assisted de novo sequencing, we identified over 90 unique spliced peptides. Splicing efficiency peaked at near-neutral pH (6.5-7.5), contrasting with classic hydrolytic profiles. Biochemical profiling revealed strong subsite selectivity, with a striking enrichment for small, aliphatic and/or hydroxyl-containing residues (Gly, Thr, Ser) at the P1 position. Furthermore, splicing preferentially targeted flexible, intrinsically disordered protein regions, with 81% of fibrinogen splicing events clustering within its C domain. In silico binding predictions for the RA-susceptibility allele HLA-DRB1*04:01 harboring the shared epitope revealed that numerous hCatK-generated spliced peptides exhibit predicted affinities exceeding those of established immunogenic and genomic sequences, uncovering protease-mediated transpeptidation as a novel post-translational modification capable of generating potent MHC class II autoantigens in RA.

biochemistry↗

bioRxiv2025

Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates

The SARS-CoV-2 main protease (3CLpro) is essential for viral replication and a leading antiviral target. Circulating variants accumulate substitutions on this enzyme, distant from the catalytic site. Surprisingly, mutant enzymes retain full proteolytic activity, though preserved overall activity does not exclude subtler effects on substrate recognition or selectivity, arising from distal structural perturbations. In this study, we compared the steady-state kinetics of wild-type (Wuhan) 3CLpro with enzymes from the Beta (K90R), Lambda (G15S), and Omicron (P132H) variants, using two peptide substrates representing distinct viral polyprotein cleavage sites. All four proteases displayed comparable catalytic efficiencies, similar pH-rate profiles, suggesting conservation of the catalytic mechanism despite sequence variation. The crystal structure of Omicron 3CLpro bound to an Nsp8-Nsp9 peptide revealed a conserved fold and active-site geometry, with the P132H side chain adopting a substrate-dependent conformation that rebuilt its local contacts, indicating towards how a distal substitution is accommodated without perturbing catalysis. Thermal stability measurements identified the sole distinguishing effect of P132H, with Omicron showing altered stability at elevated temperature. A screen of 31 tanshinones against 3CLpro identified T06 with Ki values of 5 uM, as 3CL pro inhibitor. Thus, 3CLpro may tolerate distal substitutions through local structural adaptation, supporting its durability as an antiviral target.

biochemistry↗

bioRxiv2025

Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 {+/-} 0.1 {micro}M). A 1.9 [A] crystal structure of the hCatK-Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.

biochemistry↗