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 α and seven ß 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 ß1, ß2 and ß5 subunits, while CP-17 binds the ß2 and ß5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8Å 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.

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

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 ß5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of ß5 plus either ß1 or ß2 results in improved potency.

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vent systems with prevailing extreme thermal conditions for life offer unique habitats to source heat tolearant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain , prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid- Ocean Ridges. By sequence comparisons against the MEROPS-MPRO database, globupain showed highest sequence identity to C11-like proteases present in human gut and intestinal bacteria,. Successful recombinant expression in Escherichia coli of the active zymogen and 13 mutant substitution variants allowed assesment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca²âº. When activated, the 52 kDa proenzyme was processed at Lys 137 and Lys 144 into a 12 kDa light- and 32 kDa heavy chain heterodimer. A structurally conserved His 132 /Cys 185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans . Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR- aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (T m activated enzyme = 94.51 ± 0.09°C) with optimal activity at 75 °C and pH 7.1. By characterizing globupain, our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases have been expanded. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Multiplex substrate profiling by mass spectrometry for proteases.

Proteolysis is a central regulator of many biological pathways and the study of proteases has had a significant impact on our understanding of both native biology and disease. Proteases are key regulators of infectious disease and misregulated proteolysis in humans contributes to a variety of maladies, including cardiovascular disease, neurodegeneration, inflammatory diseases, and cancer. Central to understanding a protease's biological role, is characterizing its substrate specificity. This chapter will facilitate the characterization of individual proteases and complex, heterogeneous proteolytic mixtures and provide examples of the breadth of applications that leverage the characterization of misregulated proteolysis. Here we present the protocol of Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), a functional assay that quantitatively characterizes proteolysis using a synthetic library of physiochemically diverse, model peptide substrates, and mass spectrometry. We present a detailed protocol as well as examples of the use of MSP-MS for the study of disease states, for the development of diagnostic and prognostic tests, for the generation of tool compounds, and for the development of protease-targeted drugs.