Expanding redox-based ligandability in the human cysteinome
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jing Yang
PROVIDER: PXD029761 | iProX | Tue Nov 16 00:00:00 GMT 2021
REPOSITORIES: iProX
Similar Datasets
Project description:Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.
2020-07-30 | GSE137756 | GEO
Project description:Cancer genomes are rife with genetic variants; one key outcome of this variation is gain-of-cysteine, which is the most frequently acquired amino acid due to missense variants in COSMIC. Acquired cysteines are also both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain uncharacterized. Here, we pair cysteine chemoproteomics—a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues—with genomics to reveal the hidden landscape of cysteine acquisition. For both cancer and healthy genomes, we find that cysteine acquisition is a ubiquitous consequence of genetic variation that is further elevated in the context of decreased DNA repair. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized two-stage false detection rate (FDR) error control -based FragPipe-enabled proteomic search, further enabled with a user-friendly FragPipe interface. By deploying chemoproteogenomics across 11 total cell lines, we identify gain of cysteines, including those liganded by electrophilic druglike molecules. Reference cysteines proximal to missense variants were also found to be pervasive, supporting further heretofore untapped opportunities for proteoform-specific chemical probe development campaigns. Many of the identified acquired cysteines, particularly those in mismatch repair deficient (dMMR) cell lines, were found to be predicted to be highly deleterious, providing evidence for likely functional and therapeutic relevance. The sample-matched combinatorial variant databases built into chemoproteogenomics afforded enhanced coverage and enabled identification of highly reactive and ligandable cysteine residues in the highly polymorphic MHC-Class I complexes, including for pathogenic alleles.
2024-10-03 | PXD043879 | Pride
Project description:Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of peptide-centric chemoproteomics, 1,537 S-sulfenylated sites were mapped on more than 1,000 proteins in Arabidopsis thaliana cells. The H2O2 sensitivity was quantified of more than 70% of these endogenous oxidation events toward exogenous H2O2 stimulation. Proteins involved in RNA and metabolic processing were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located in catalytic sites of enzymes or on cysteines involved in metal binding, hinting at direct mode-of-actions for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which is speculated to be a redox-regulatory site. Replacement of the noncatalytic Cys181 of the recombinant AtMAPK4 by the redox-insensitive serine decreased the protein kinase activity, emphasizing the importance of this noncatalytic cysteine. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis plants under oxidative stress and delivered an unprecedented inventory for unraveling the precise role of these oxidized cysteines in plant redox signaling.
2019-09-16 | PXD013495 | Pride
Project description:Cell surface cysteines represent an attractive class of residues for chemoproteomic studies due to their accessibility towards drugs and key roles they play in the structure and function of most human proteins. The redox sensitivity of cysteines also makes cell surface cysteines potential markers for cellular activities with altered redox states. However, cell surface cysteines are underrepresented in most of the cysteine proteomic dataset. While many mass spectrometry (MS) based techniques have been developed to enrich membrane proteins, current studies lack methods that can specifically inventory cysteines on cell surfaces. Here, we developed a novel dual enrichment method to achieve chemoproteomic profiling of cell Surface Cysteines - “Cys-Surf''. Combining cell surface capture (CSC) biotinylation and cysteine chemoproteomic biotinylation, this two-step biotinylation platform achieves identification of more than 1,900 cysteines with a specificity of about 50.0% in cell surface localization. In addition, Cys-Surf achieved quantitative analysis of oxidation states of cell surface cysteines, which reflect a completely different profile compared to that of the whole cysteinome. Redox sensitive cell surface cysteines were identified by applying reducing reagents and during T cell activation. Cys-Surf is also compatible with competitive small-molecule screening by isotopic tandem orthogonal activity-based protein profiling (isoTOP-ABPP) to evaluate the ligandability of cell surface cysteines. Altogether, these findings establish a platform that enables redox and ligandability analysis of the cell surface cysteinome and sheds light on future functional studies and drug discovery efforts targeting cell surface cysteines.
2024-01-26 | PXD042403 | Pride
Project description: Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human 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 cancer cell proliferation. The resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in >110 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
2023-09-06 | PXD041314 | Pride
Project description:Cysteine residues undergo various oxidative modifications, acting as key sensors of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Given that ROS and RNS have known roles in many pathophysiological processes, numerous proteome-wide strategies to profile cysteine oxidation state have emerged in the last decade. Recent advancements to traditional redox profiling methods include incorporation of costly isotopic labeling reagents to allow for more quantitative assessment of oxidation states. These methods are typically carried out by using sequential thiol capping and reduction steps in order to label redox-sensitive cysteines, often found in di-cysteine motifs (‘CXXC’ or ‘CXXXC’). Tailored, pricy algorithms are commonly used to analyze redox-profiling datasets, the majority of which cannot accurately quantify site-of-labeling in redox-motifs; moreover, accurate quantification is confounded by excess labeling reagents during sample preparation. Here, we present a low-cost redox-profiling workflow using newly synthesized isotopic reagents compatible with SP3-bead technology, termed SP3-ROx, that allows for high throughput, rapid identification of redox-sensitive cysteines. We optimize cysteine labeling quantification using the FragPipe suite, an open source GUI for MSfragger-based search algorithm. Application of SP3-ROx to naive and activated T cells identifies redox-senstive cysteines, showcasing the utility of this workflow to study biological processes.
2022-03-02 | PXD029500 | Pride
Project description:Cysteine residues undergo various oxidative modifications, acting as key sensors of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Given that ROS and RNS have known roles in many pathophysiological processes, numerous proteome-wide strategies to profile cysteine oxidation state have emerged in the last decade. Recent advancements to traditional redox profiling methods include incorporation of costly isotopic labeling reagents to allow for more quantitative assessment of oxidation states. These methods are typically carried out by using sequential thiol capping and reduction steps in order to label redox-sensitive cysteines, often found in di-cysteine motifs (‘CXXC’ or ‘CXXXC’). Tailored, pricy algorithms are commonly used to analyze redox-profiling datasets, the majority of which cannot accurately quantify site-of-labeling in redox-motifs; moreover, accurate quantification is confounded by excess labeling reagents during sample preparation. Here, we present a low-cost redox-profiling workflow using newly synthesized isotopic reagents compatible with SP3-bead technology, termed SP3-ROx, that allows for high throughput, rapid identification of redox-sensitive cysteines. We optimize cysteine labeling quantification using the FragPipe suite, an open source GUI for MSfragger-based search algorithm. Application of SP3-ROx to naive and activated T cells identifies redox-senstive cysteines, showcasing the utility of this workflow to study biological processes.
2022-03-02 | PXD031647 | Pride
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.
2023-09-06 | PXD038232 | Pride
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.
2023-09-06 | PXD038239 | Pride
Project description:Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.
2024-02-12 | PXD049322 | Pride