Project description:Current methods for measuring amino-acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts, and software to boost data acquisition in real-time on the mass spectrometer. Our method, Streamlined Cysteine Activity-Based Protein Profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites in 18 min per compound. We applied it to identifying the proteome-wide targets of a covalent inhibitor of mutant KRASG12C and of ibrutinib, a covalent inhibitor of BTK. In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines , which includes >20,000 cysteines in >6,000 proteins per cell line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
Project description:Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is associated with enhanced expression during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. When added to cells, LN5P45 shows outstanding target engagement and proteome-wide selectivity. Importantly, we found that LN5P45 and the other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 via lysine 31. Thus, our findings provide novel insights for the design of future OTUB2-related therapeutics and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
Project description:Chemoproteomics investigates small molecule-protein interactions and has made significant progress in recent years. Despite its vast potential, the proteome-wide profiling of reactive cysteine ligandability remains a formidable task to adapt for high throughput applications. This is primarily due to a lack of platforms capable of achieving the desired depth using low sample input in 96- or 384-well plates. Here we have revamped the cysteine profiling platform to address the challenge with an eye toward performing high-throughput library screening in plates. By incorporating several changes including i) an 18-plex TMT sample multiplexing strategy, ii) a magnetic beads-based one-pot workflow, iii) a 10X higher capacity streptavidin resin, and iv) optimized mass spectrometry analyses, a plate-based platform was developed that enables routine interrogation of either ~18,000 or ~24,000 reactive cysteines based on starting amounts of 10 or 20 µg, respectively. We applied the platform to screen a library of 192 electrophiles in the native HEK293T proteome, mapping the ligandablity of 38,450 reactive cysteines from 8,274 human proteins. The significantly improved depth revealed many previously unknown reactive cysteines and cysteine-ligand interactions and led to the identification of an azepane-containing acrylamide which has preferential binding to cysteines in EGF-like domains. We further applied the platform to characterize new cellular targets of well-studied compounds and covalent drugs in three different human cell lines. We found that ARS-1620, a KRASG12C inhibitor, also binds to cysteine 140 of an off-target adenosine kinase ADK, inhibiting its kinase activity. The platform represents a major step forward to high throughput evaluation of reactive cysteines on a proteome-wide scale.
Project description:Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFkB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting,and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity
Project description:Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract (ARNC). To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- vs. intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ~4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, while γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33 and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130 and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose C42, C79/80, C154 in γC; C42, C33 in γD, and C83, C115 and C130 in γS. In conclusion, while the β-crystallin disulfidome is highly conserved in ARNC and LEGSKO mouse, and reproducible by in vitro oxidation, some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of ARNC.
Project description:H. jecorina undergoes a heterothallic reproductive cycle, and the mating yields asci with 16 linearly arranged ascospores. The sixteen ascospores are generated via two rounds of postmeiotic mitosis following the two meiotic divisions. We have discovered that viable aneuploid ascosporesare are frequently (~90%) generated due to a net gain of a conserved ~500 kbp genomic segment. Here we have compared whole genome gene copy number changes in the aneuploid F1 progeny, euploid F1 progeny, parental strains and the F1 progeny of RTU strain backcross with CBS999.97 . We determined genome-wide gene copy number in the genomes of ascospores generated from haploid wild-type strain. We also determined genome-wide gene copy number in the genomes of the F1 progeny of CBS999.97 wild type.
Project description:Array-based comparative genome hybridization. The aneuploid progeny could return to euploid state. We determined genome-wide gene copy number in the genomes of aneuploid progeny after 14, 24 and 34 days vegetative propagation in dextrose-containing rich medim.