Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis.

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified ("dark peptidome") by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.

Discovery of small-molecule enzyme activators by activity-based protein profiling.

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

Comparability of Objective Structured Clinical Examinations (OSCEs) and Written Tests for Assessing Medical School Students' Competencies: A Scoping Review.

Objective Structured Clinical Examinations (OSCEs) and written tests are commonly used to assess health professional students, but it remains unclear whether the additional human resources and expenses required for OSCEs, both in-person and online, are worthwhile for assessing competencies. This scoping review summarized literature identified by searching MEDLINE and EMBASE comparing 1) OSCEs and written tests and 2) in-person and online OSCEs, for assessing health professional trainees' competencies. For Q1, 21 studies satisfied inclusion criteria. The most examined health profession was medical trainees (19, 90.5%), the comparison was most frequently OSCEs versus multiple-choice questions (MCQs) (18, 85.7%), and 18 (87.5%) examined the same competency domain. Most (77.5%) total score correlation coefficients between testing methods were weak (r < 0.40). For Q2, 13 articles were included. In-person and online OSCEs were most used for medical trainees (9, 69.2%), checklists were the most prevalent evaluation scheme (7, 63.6%), and 14/17 overall score comparisons were not statistically significantly different. Generally low correlations exist between MCQ and OSCE scores, providing insufficient evidence as to whether OSCEs provide sufficient value to be worth their additional cost. Online OSCEs may be a viable alternative to in-person OSCEs for certain competencies where technical challenges can be met.

Integrative X-ray Structure and Molecular Modeling for the Rationalization of Procaspase-8 Inhibitor Potency and Selectivity.

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ X-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

A Commensal Dipeptidyl Aminopeptidase with Specificity for N-Terminal Glycine Degrades Human-Produced Antimicrobial Peptides in Vitro.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Indole-3-carbinol (I3C) analogues are potent small molecule inhibitors of NEDD4-1 ubiquitin ligase activity that disrupt proliferation of human melanoma cells.

The HECT domain-containing E3 ubiquitin ligase NEDD4-1 (Neural precursor cell Expressed Developmentally Down regulated gene 4-1) is frequently overexpressed in human cancers and displays oncogenic-like properties through the ubiquitin-dependent regulation of multiple protein substrates. However, little is known about small molecule enzymatic inhibitors of HECT domain-containing ubiquitin ligases. We now demonstrate that indole-3-carbinol (I3C), a natural anti-cancer phytochemical derived from cruciferous vegetables such as cabbage and broccoli, represents a new chemical scaffold of small molecule enzymatic inhibitors of NEDD4-1. Using in vitro ubiquitination assays, I3C, its stable synthetic derivative 1-benzyl-I3C and five novel synthetic analogues were shown to directly inhibit NEDD4-1 ubiquitination activity. Compared to I3C, which has an IC50 of 284µM, 1-benzyl-I3C was a significantly more potent NEDD4-1 enzymatic inhibitor with an IC50 of 12.3µM. Compounds 2242 and 2243, the two indolecarbinol analogues with added methyl groups that results in a more nucleophilic benzene ring π system, further enhanced potency with IC50s of 2.71µM and 7.59µM, respectively. Protein thermal shift assays that assess small ligand binding, in combination with in silico binding simulations with the crystallographic structure of NEDD4-1, showed that each of the indolecarbinol compounds bind to the purified catalytic HECT domain of NEDD4-1. The indolecarbinol compounds inhibited human melanoma cell proliferation in a manner that generally correlated with their effectiveness as NEDD4-1 enzymatic inhibitors. Taken together, we propose that I3C analogues represent a novel set of anti-cancer compounds for treatment of human melanomas and other cancers that express indolecarbinol-sensitive target enzymes.