Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-D-glycero-α-D-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is D-glycero-L-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-D-glycero-α-Dmanno-heptose to GDP-D-glycero-4-keto-α-D-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-D-glycero-4-keto-β-L-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-D-glycero-β-L-gluco-heptose. The threedimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-D-glycero-β-L-glucoheptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/ reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.
Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.
We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3K GOF/+). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3K GOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF- pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3K GOF/+. Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF- signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction.
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