Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA260045 | ENA |
REPOSITORIES: ENA
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Project description:Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing the appropriate E3 ligase for a certain targeted protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, it is well known that due to the large sizes of PROTAC molecules, their cellular uptake level remains an issue, posing a challenge to translate PROTACs into therapeutics. Driven by our fundamental investigation to compare how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, may affect the degradation of a model protein Bruton's Tyrosine Kinase (BTK), we serendipitously discovered that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular concentration and target engagement of the PROTAC. Building on this discovery, we developed RC-1 as the first reversible covalent BTK PROTAC, which has high target occupancy and is effective as both an inhibitor and a degrader. Molecular dynamics calculations and phase-separation based ternary complex assays support that RC-1 forms a stable ternary complex with BTK and Cereblon (CRBN). Additionally, RC-1 compares favorably with other reported BTK degraders in cell viability and target engagement assays and has a reasonable plasma half-life for in vivo applications. Importantly, this reversible covalent strategy can be generalized and applied to improve other PROTACs. This work can not only help to develop optimal BTK degraders for clinical applications but also provide a new strategy to improve PROTAC efficacy.
2020-02-04 | MSV000084902 | MassIVE
Project description:We have developed a covalent linkage-based SpyCLIP method by taking advantage of SpyTag-SpyCatcher chemistry, which circumvents gel purification steps of captured RBP-RNA needed for all existing CLIP methods, thereby dramatically reducing the technical difficulty and enabling high-throughput application of the CLIP technology.
2019-01-21 | GSE114720 | GEO
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.
2015-09-03 | GSE60988 | GEO
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments
2015-09-03 | E-GEOD-60988 | biostudies-arrayexpress
Project description:We previously identified functional distinct sub-populations in adult male gWAT Pdgfrb+ cells utilizing the MuralChaser lineage tracking system and single-cell RNA-seq. Later in this related submission, we performed single-cell RNA-seqs on different developing stage of gWAT (P3, P7 and 5-week). We identified 4 sub-populations of Pdgfrb+ cells. Bulk-mRNA were conducted to these sub-populations in order to understand their transcriptomic profiles.
2021-08-23 | GSE181245 | GEO
Project description:Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here we devised Modifications, Interactions and Phenotypes by Affinity Purification Mass Spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependecies between covalent, reversible and functional protein complex regulations by knock-out or site-specific mutation, revealed isgylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p-38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.
2021-07-07 | PXD010996 | Pride
Project description:Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.
2018-07-19 | GSE110951 | GEO
Project description:Effective and convenient covalent protein conjugation could benefit diverse biological systems, stabilizing key protein protein interactions which are otherwise reversible. Neisseria meningitidis contains a protein which undergoes autoproteolysis via an anhydride. Here, we harness this spontaneously generated electrophile for covalent targeting of unmodified endogenous proteins. In ‘NeissLock’, a binding protein genetically fused to the self-processing module (SPM) docks to its target protein. Upon triggering with calcium, the aspartic anhydride generated at the C-terminus of the binding protein allows nucleophilic attack by nearby residues on the target protein, so ligating the proteins. We established a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We validated and optimized the NeissLock concept using the Ornithine Decarboxylase/Antizyme complex. A range of nucleophiles on the target (α-amine or ε-amines) could rapidly react with the anhydride, but reaction was blocked if the partner protein did not dock. Surprisingly, the optimal pH for covalent ligation was 7.0. We then armed Transforming Growth Factor-α with SPM and established covalent targeting to Epidermal Growth Factor Receptor at the surface of living cells. NeissLock harnesses exceptional protein chemistry to allow covalent targeting of endogenous proteins under mild conditions with up to 80% yield, allowing new possibilities for molecular engineering.
2021-02-01 | PXD023073 | Pride
Project description:ALARM MSPS files for identification of reversible covalent inhibitors.
2020-07-20 | MSV000085781 | MassIVE
Project description:More than half of the ∼20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.
2024-09-03 | PXD053077 | Pride