Biology subjects

Hurysz, B.

Publications and source records attributed to Hurysz, B..

3 recordsLinked to original sources

bioRxiv2025

mspms: A Comprehensive R Package and Graphic Interface for Multiplex Substrate Profiling by Mass Spectrometry Analysis

Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) is a powerful method for determining the substrate specificity of proteolytic enzymes, knowledge key for developing protease inhibitors, diagnostics, and protease-activated therapeutics. However, the complex datasets generated by MSP-MS pose significant analytical challenges. To address this, we developed mspms, a Bioconductor R package complemented by an intuitive graphical interface. Mspms streamlines MSP-MS data analysis by standardizing workflows for data preparation, processing, statistical analysis, and visualization. Designed for accessibility, it serves both advanced users via the R package and broader audiences through the web interface. We validated mspms by profiling the substrate specificity of four well-characterized cathepsins (A-D), demonstrating its ability to reliably capture expected substrate specificities. As the first publicly available platform for MSP-MS data analysis, mspms delivers comprehensive functionality, transparency, and ease of use, making it a valuable resource for the protease research community. Access to mspms is available through the Bioconductor project at https://bioconductor.org/packages/mspms, and a graphic interface is available at https://gonzalezlab.shinyapps.io/mspms_shiny/. Author SummaryWe developed mspms, an easy-to-use tool that helps researchers analyze data from a proteomics technique called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). This software improves on previous methods of analyzing MSP-MS data, which required the user to navigate a confusing mix of R scripts, manual manipulation of spreadsheets, and third-party tools--an approach that was daunting for collaborators and new graduate students alike. Mspms streamlines the process, enabling faster, more reliable, and reproducible data analysis. We tested the tool using well-known proteases and found that it accurately identifies their known targets. As the first comprehensive tool for MSP-MS analysis, mspms makes this method approachable to a wider audience. Its available for free through the Bioconductor project at https://bioconductor.org/packages/mspms, and a graphical interface is available at https://gonzalezlab.shinyapps.io/mspms_shiny/.

bioinformatics↗

bioRxiv2023

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

Proteasomes are essential for protein homeostasis in mammalian cells1-4 and in protozoan parasites such as Trichomonas vaginalis (Tv).5 Tv and other protozoan 20S proteasomes have been validated as druggable targets.6-8 However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven and seven {beta} subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB)9 and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the {beta}1, {beta}2 and {beta}5 subunits, while CP-17 binds the {beta}2 and {beta}5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8[A] resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

biophysics↗

bioRxiv2023

Development of subunit selective substrates for Trichomonas vaginalis proteasome

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the {beta}5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of {beta}5 plus either {beta}1 or {beta}2 results in improved potency.

biochemistry↗