The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

Center for Discovery and Innovation in Parasitic Diseases Skaggs School of Pharmacy and pharmaceutical Sciences University of California San Diego La Jolla CA USA

Department of Medicine School of Medicine University of California San Diego La Jolla CA USA

Department Pharmaceutical Sciences College of Pharmacy The University of Jordan Amman Jordan

Institute of Organic Chemistry and Biochemistry AS CR v v i Prague Czech Republic

Scripps Institution of Oceanography University of California San Diego La Jolla CA USA

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA USA

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bioRxiv. 2023 Aug 17:2023.08.17.553660. doi: 10.1101/2023.08.17.553660. PubMed

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A recombinant expression system for the Plasmodium falciparum proteasome enables structural analysis of its assembly and the design of selective inhibitors

Structural Insights into Salinosporamide a Mediated Inhibition of the Human 20S Proteasome

Structural insights into Salinosporamide A mediated inhibition of the human 20S proteasome

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PDB
8OIX, 8P0T, 4R3O, 5LF3, 7AWE, 7PG9, 7ZYJ, 8OIX, 8P0T