Project description:Suzuki-Miyauara cross-coupling has been used for mass-spectrometry-based chemoproteomics, including for target deconvolution and site of labeling studies. We also demonstrated that Suzuki-Miyauara cross coupling and CuAAC can be combined for dual target labeling for mass spectrometry-based proteomics. Furthermore, multiplexed CuAAC Suzuki-Miyaura chemoproteomics platform has been successfully applied to identify the protein targets of bifunctional cysteine- and lysine-reactive crosslinking probes.
Project description:Adaptor proteins often serve as hubs to regulate diverse protein complexes in cells. This multitude of functions can complicate the study of adaptors, as their genetic disruption typically impairs the activities of all parent complexes (or ‘complexoforms’). Here we describe the chemical proteomic analysis of bicyclopyrrolidine acrylamide stereoprobes in human cells, leading to the discovery of a ligand – FWG-33B – that stereoselectively reacts with cysteine-100 (C100) of the methyltransferase (MT) adaptor TRMT112. FWG-33B showed negligible reactivity with uncomplexed recombinant TRMT112, and this interaction was restored exclusively in the presence of METTL5, but not other MT partners of TRMT112. A co-crystal structure revealed that FWG-33B binds at a composite pocket proximal to C100 that is templated by METTL5 and absent in other TRMT112:MT complexes. Subtle structural rearrangements promoted by FWG-33B in turn lead to allosteric agonism of METTL5, thus revealing how covalent ligands targeting a pleiotropic adaptor can confer partner-specific functional effects through reactivity with a single complexoform.
Project description:Activity-based protein profiling has identified hundreds of proteins from diverse classes that react site-specifically with stereo-defined electrophilic compounds (stereoprobes) in human cells. The structure-activity relationships underlying these stereoprobe-protein interactions, however, remain poorly understood. Here we show that the protein interaction landscape of tryptoline acrylamide stereoprobes can be profoundly altered by structural modifications distal to the acrylamide reactive group. Stereoprobe liganding events, many of which occurred at non-orthosteric sites, mostly evaded assignment by affinity prediction models (e.g., Boltz-2), which instead tended to redirect the ligands to orthosteric pockets (an outcome we refer to as “orthostery burnout”). We discovered that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial Hsp70 chaperone (mortalin/HSPA9), leading to impairments in mitochondrial protein import and induction of mitophagy. Our results highlight tryptoline acrylamides as a versatile source of first-in-class covalent ligands targeting non-orthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.
Project description:We utilized the well-characterized murine T cell transfer model of colitis to find specific alterations in the intestinal luminal proteome associated with inflammation. Mass spectrometry proteomic analysis of colonic samples permitted the identification of ~10,000-12,000 unique peptides that corresponded to 5610 protein clusters identified across three groups, including the colitic Rag1 -/- T cell recipients, isogenic Rag1 -/- controls, as well as wild-type mice. Bioinformatic analyses on host and microbial proteins found specific proteins and GO term functionalities unique to each group, as well as GO terms shared across the three cohorts. We further demonstrated that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia that was substantiated with 16S rDNA sequencing.
Project description:Transcriptional regulatory proteins are frequent drivers of oncogenesis and common targets for drug discovery. The transcriptional co-activator, ENL, which binds to chromatin through an acetyllysine reader YEATS domain, is preferentially required for the survival and pathogenesis of acute leukemia. Small molecules that inhibit the ENL YEATS domain show anti-leukemia effects in preclinical models, which is thought to be caused by the downregulation of pro-leukemic ENL target genes. However, the transcriptional effects of ENL YEATS domain inhibitors have not been studied in models of intrinsic or acquired resistance and, therefore, the connection between proximal transcriptional effects and downstream anti-proliferative effects is poorly understood. To address this, we identified models of intrinsic and acquired resistance and used them to study the effects of ENL YEATS domain inhibitors. We first discovered that ENL YEATS domain inhibition produces similar transcriptional responses in naive models of sensitive and resistant leukemia. We then performed a CRISPR/Cas9-based genetic modifier screen and identified in-frame deletions of the essential transcriptional regulator, PAF1, that confer resistance to ENL YEATS domain inhibitors. Using isogenic models of PAF1-mediated resistance, we again found that the downregulation of ENL target genes is shared in both sensitive and resistant leukemia. Altogether, these data support the conclusion that the suppression of ENL target genes is not sufficient to explain the anti-leukemia effects of ENL antagonists.
Project description:Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We finally demonstrate how measurements of reactivity in native versus denatured proteomes can further discriminate essential cysteines amendable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
Project description:Recent studies demonstrated that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts induced augmented glycolysis and production of alpha smooth muscle actin (αSMA) and collagen1. Knockout of COUP-TFII decreased glycolysis and collagen1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.
Project description:Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.
Project description:Established bacterial proteome sample preparations, including sonication and bead beating, leave insoluble carbohydrate-rich cell envelope pellets with an abundance of vital proteins often overlooked or missed in LC-MS/MS analyses. Triflic acid selectively removes glycans and we demonstrate that in comparison to sonication alone, incubation of whole bacterial cells as well as post-sonication insoluble pellets yields membrane and cell envelope-associated proteins for LC-MS/MS detection. We provide a detailed side-by-side comparison of triflic acid and sonication preparations for Gram- (Pseudomonas aeruginosa), Gram+ (Bacillus subtilis), and a complex bacterial human distal gut microbiome sample. Further, human Jurkat cells that lack a peptidoglycan and are readily solubilized by established methods, reveal only subtle differences in measurable proteins by LC-MS/MS between sonication and triflic acid preparations. Critically, we show that our new triflic acid-based proteome preparation method is broadly applicable and greatly improves our ability to detect and quantitate bacterial cell envelope proteins.
Project description:Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across the two mouse cohorts, as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control; thereby, limiting genetic and environmental variables that influence microbiome composition.