Search

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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

Here we report AWP28, an activity-based probe that can be used to biochemically monitor caspase-1 activation in response to proinflammatory stimuli. Using AWP28, we show that apoptosis is triggered upon Salmonella enterica var. Typhimurium infection in primary mouse bone marrow macrophages lacking caspase-1. Furthermore, we report that upon Salmonella infection, inflammasome-mediated caspase-1 activity is required to bypass apoptosis in favor of proinflammatory pyroptotic cell death.

MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.

Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.