cathepsin l activity assay kit bps bioscience  (BPS Bioscience)

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A ) Schematic of in-silico docking screen workflow. A panel of 48 respiratory proteases and 10 SERPINs upregulated upon viral infection were assembled. 3D structures were generated, with annotated protease active sites and SERPIN reactive center loops (RCLs). SERPIN-protease docking was performed using HADDOCK, guiding the RCL into the protease active site, and obtaining raw HADDOCK scores to reflect predicted binding energies. The dataset was normalized to the mean, and thresholds set for high-confidence in-silico binders (mean of positive controls) and high-confidence in-silico non-binders (mean of negative controls). Top-scoring complexes of interest were visually assessed for 3D RCL fit, exosite formation, and the positioning of protease active residues relative to SERPIN P4-P4’ residues. ( B ) Heatmap of docking results, with z-scores centered to the mean of control SERPIN-protease pairs and normalized for each SERPIN. The darker the red, the more favorable the binding energies. LEI leukocyte elastase inhibitor encoded by SERPINB1 , PAI-2 plasminogen activator inhibitor 2 encoded by SERPINB2 , Leupin encoded by SERPINB4 , PI-8 protease inhibitor 8 encoded by SERPINB8 , CAP-3 cytoplasmic anti-protease 3 encoded by SERPINB9 , Bomapin encoded by SERPINB10 , Headpin encoded by SERPINB13, ATIII antithrombin 3 encoded by SERPINC1, PAI-1 plasminogen activator inhibitor 1 encoded by SERPINE1, C1-INH C1 inhibitor encoded by SERPING1 . V validated pairs in vitro as shown in Fig. . ( C ) Normalized HADDOCK scores of PAI-1 with its positive controls (thrombin, active TMPRSS2, trypsin, kallikrein, uPA), negative controls (furin, uPA to PAI-1 P4-P4’ alanine substitutions, and uPA to PAI-1 P1 mutant R346A), the TMPRSS2 zymogen (TMPRSS2-z), and cathepsins. Green dotted line (mean of all positive controls in the in-silico screen) is the threshold for high-confidence binders; red dotted line (mean of all negative controls in the in-silico screen) is the threshold for high-confidence non-binders. Symbols: triangles, trypsin-like proteases; circles, subtilisin-like proteases; split circle, thrombin; hexagon, kallikreins; diamonds, cathepsins; light blue inactive serine protease, inactive; blue, serine proteases; yellow, cysteine proteases; salmon, aspartic proteases. Blue, serine proteases; yellow, cysteine proteases; red, aspartic proteases. ( D – F ) Docking structures of top-scoring complexes for PAI-1 with TMPRSS2 (zymogen), PAI-1 with TMPRSS2 (active) and CTSL, respectively. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: In Silico, Infection, Generated, Binding Assay, Control, Protease Inhibitor, In Vitro, Mutagenesis

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A , B ) Normalized reaction rates for uPA, TMPRSS2, cathepsin L, and cathepsin B in the absence or presence of PAI-1 ( A ), or PAI-2 ( B ) from in vitro fluorometric assays. Statistical analysis by One-way ANOVA and Kruskal–Wallis test, * P < 0.05. Exact P values in ( A ). uPA, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; TMPRSS2, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; CTSL, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; CTSB, 0 vs. 0.025 nM P > 0.9999, 0 vs. 0.25 nM P = 0.5934. Exact P values in ( B ). uPA, 0 vs. 1 nM P = 0.3594, 0 vs. 10 nM P = 0.0146; TMPRSS2, 0 vs. 1 nM P > 0.9999, 0 vs. 10 nM P = 0.9121; CTSL, 0 vs. 1 nM P > 0.9999, 0 vs. 10 nM P = 0.0722; CTSB, 0 vs. 1 nM P = 0.3594, 0 vs. 10 nM P = 0.0146. Mean ± SD, n = 3 replicates. Raw data in Dataset EV . ( C ) SDS-PAGE and silver stain of mixed recombinant active uPA (32 kDa, not visible) and PAI-1 (43 kDa). ( D ) SDS-PAGE and silver stain of mixed recombinant active TMPRSS2 (31 kDa) and PAI-1 (43 kDa). ( E ) SDS-PAGE and silver stain of mixed recombinant cathepsin L (32 kDa) and PAI-1 (43 kDa). *Denotes use of PAI-1 inhibitor triplaxinin and PAI-1 cleavage products. (F) SDS-PAGE and silver stain of mixed recombinant cathepsin L with PAI-1, reactions were conducted at pH 6.5 and pH 5.5. ( G ) SDS-PAGE and silver stain of mixed recombinant cathepsin B with PAI-1. ( H ) Schematic of importance of activity of TMPRSS2 and cathepsin L in SARS-CoV-2 entry by lineage. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: In Vitro, SDS Page, Silver Staining, Recombinant, Activity Assay

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A – C ) Schematic of fit of substrate residues into protease pockets for uPA, TMPRSS2, and cathepsin L, respectively. Substrates include zymogen self-cleavage region, canonical or previously described substrate, PAI-1 and fluorophore-linked peptide substrate used in this study. Amino acids are colored according to their side chain chemistry (Unipro UGENE): basic (R, K) litmus blue with R being more basic and darker; acidic (E, D) litmus red with more acidic being darker; hydrophobic (I, L, V, A), yellow with intensity corresponding to hydrophobic character; sulfur-containing (C, M) green; aromatic (F, Y, W) in teal; polar (N, Q, S, T) magenta/pink with darker coloring for more polarity; non-polar glycine (G) in dark gray and proline (P) in light gray. Assembled from references (Koga et al, ; Menard et al, ; Rossignol et al, ; Shrimp et al, ) and UNIPROT.

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques:

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A – D ) SARS-CoV-2 WA-1 ( A , B ) or Omicron BA.1 ( C , D ) multi-cycle infection in Calu-3 cells with extracellular addition of buffer, 12.5 or 25 ng/mL of active PAI-1 or of 25 ng/mL heat-inactivated (HI) PAI-1 (48 hpi); or 10 or 100 ng/mL anti-PAI-1 depleting antibody or 10 ng/mL isotype IgG control (48 hpi) by high-content microscopy. Statistical analysis by One-way ANOVA and Holm-Sidak’s multiple comparison test, ** P < 0.005. Exact P values in ( A ). buffer vs. HI rPAI-1 P = 0.9407; buffer vs. rPAI-1 P < 0.0001; anti-PAI-1 (1:100) vs. IgG control P = 0.0059; anti-PAI-1 (1:50) vs. IgG control P < 0.0001. Exact P values in ( C ). buffer vs. HI rPAI-1 P = 0.5441; buffer vs. rPAI-1 P < 0.0001; IgG vs. anti-PAI-1 (1:100) P = 0.7968; IgG vs. anti-PAI-1 (1:50) P = 0.1473. n = 6 biological replicates. Representative images, DAPI (blue, nuclei), SARS-CoV-2 nucleoprotein (red). ( E ) Extracellular titers of SARS-CoV-2 WA-1 grown at low MOI-infection on Calu-3 cells for 48 h. Infectious titers were determined by TCID 50 assay on Calu-3. Statistical analysis by one-sided t test, * P < 0.05.Exact P value P = 0.05, n = 3 biological replicates ( F ). BHK cells co-transfected to express SARS-CoV-2 spike, TMPRSS2, and PAI-1. Spike S2 and 2’ band intensities by western blot and densitometry. % S2’ cleaved determined by ratio of S2’ vs S2’ + S2 band intensity from the blot shown. FL full-length. ( G ) BHK cells co-transfected to express SARS-CoV-2 spike and TMPRSS2, and rPAI-1 or buffer control added to the cell supernatant. Spike S2 and 2’ band intensities by western blot and densitometry. S2’ % cleaved determined by the ratio of S2 vs S2’ band intensity from the blot shown. FL full-length. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: Infection, Control, Microscopy, Comparison, Transfection, Western Blot