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Results for “Activity-based protein profiling”

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7 recordsLinked to original sources
PubMed2026

Functional mapping of the Trypanosoma cruzi serinome by fluorophosphonate activity-based protein profiling.

Serine hydrolases (SHs) constitute one of the largest enzyme superfamilies in eukaryotes, yet their roles in Trypanosoma cruzi, the causative agent of Chagas disease, remain largely uncharacterized. Here, we report an activity-based chemoproteomic map of the T. cruzi epimastigote serinome by combining genome-informed in silico curation with whole-cell activity-based protein profiling (ABPP) using a panel of cell-permeable fluorophosphonate (FP)-alkyne probes. Whole-cell labelling followed by label-free quantitative proteomics (LFQ-MS) identified 37 enriched SH-like proteins, including 35 with conserved or partially conserved catalytic triad/dyad features, spanning lipases, peptidases, esterases, and previously uncharacterized hydrolases. The 35 SHs represent approximately 63% of the 56 predicted SHs retained after catalytic-site curation. Domain architecture analysis revealed broad structural diversity, while orthologue-based localization data suggested association with multiple subcellular compartments, including glycosomal, mitochondrial, and endosomal localizations. Gene Ontology enrichment highlighted lipid metabolic and catabolic processes as dominant functional themes, and protein-protein interaction network analysis supported functional connectivity among the captured enzymes. Several identified SHs, including oligopeptidase B, prolyl oligopeptidase Tc80, serine carboxypeptidase CPB1, and phospholipase A1 (PLA1) have previously been characterized in trypanosomatids, with roles linked to parasite virulence or host-pathogen interactions. Together, these findings establish a fluorophosphonate-based chemoproteomic resource for the kinetoplastid community and prioritize probe-accessible active T. cruzi SHs for future functional validation and antiparasitic inhibitor discovery.

Activity-based protein profiling↗
PubMed2026

Mapping the covalent cysteine interactome of Ebselen reveals high-sensitivity target engagement and redox proteome remodeling.

Ebselen is a covalent organoselenium compound with broad pharmacological activity, yet its cellular cysteine targets and downstream proteomic consequences remain incompletely defined. Here, we integrated competitive gel-based activity-based protein profiling, reactivity-dependent tandem orthogonal proteolysis-activity-based protein profiling, and TMT-based quantitative proteomics to map Ebselen-induced cysteine engagement and proteome remodeling in living cancer cells. Ebselen exhibited dose-dependent cytotoxicity and markedly perturbed intracellular thiol-redox balance, as reflected by glutathione depletion and altered reactive oxygen species-associated fluorescence readouts. Competitive gel-based profiling confirmed concentration-dependent engagement of protein cysteine residues in live cells. Quantitative rdTOP-ABPP further identified hundreds of dose-responsive cysteine sites in HeLa and HepG2 cells and revealed a preference for cysteine microenvironments enriched with basic residues. Cross-cell-line comparison highlighted CDK5 Cys53, SMU1 Cys298, and RPSA2 Cys163 as conserved covalent nodes, among which CDK5 Cys53 showed high sensitivity to Ebselen treatment, a finding validated by competitive labeling and MS-based site assignment. Global TMT proteomics revealed extensive remodeling of redox-related and cell-survival-associated pathways, including compensatory upregulation of selenoproteins such as TXNRD1 and GPX family members. Together, these results define a chemical proteomic atlas of Ebselen-cysteine interactions and provide a framework for understanding and optimizing covalent organoselenium therapeutics.

Humans↗
PubMed2024

Chemical proteomics to study metabolism, a reductionist approach applied at the systems level.

Cellular metabolism encompasses a complex array of interconnected biochemical pathways that are required for cellular homeostasis. When dysregulated, metabolism underlies multiple human pathologies. At the heart of metabolic networks are enzymes that have been historically studied through a reductionist lens, and more recently, using high throughput approaches including genomics and proteomics. Merging these two divergent viewpoints are chemical proteomic technologies, including activity-based protein profiling, which combines chemical probes specific to distinct enzyme families or amino acid residues with proteomic analysis. This enables the study of metabolism at the network level with the precision of powerful biochemical approaches. Herein, we provide a primer on how chemical proteomic technologies custom-built for studying metabolism have unearthed fundamental principles in metabolic control. In parallel, these technologies have leap-frogged drug discovery through identification of novel targets and drug specificity. Collectively, chemical proteomics technologies appear to do the impossible: uniting systematic analysis with a reductionist approach.

Humans↗
PubMed2026

Chemoproteomics Prioritizes Mitochondrial ADP/ATP Translocase as a Candidate Target Associated with 6PPDQ-Induced Respiratory Toxicity in Rainbow Trout Gill.

6PPD quinone (6PPDQ) is an emerging contaminant that induces acute respiratory toxicity in rainbow trout (Oncorhynchus mykiss), yet its underlying molecular mechanisms remain poorly understood. In the present study, short-term in vivo exposure of rainbow trout to 6PPDQ resulted in substantial accumulation and limited biotransformation of 6PPDQ in the gill, accompanied by pronounced gill structural damage and increased whole-fish oxygen consumption. Taking advantage of the electrophilic reactivity of the quinone moiety of 6PPDQ toward cysteine residues, we applied activity-based protein profiling (ABPP) to gill tissue. ABPP revealed marked alterations in mitochondrial cysteine reactivity and highlighted ADP/ATP translocase (ANT) as a candidate 6PPDQ-interacting mitochondrial protein. A Cys-160-containing ANT peptide within the nucleotide-binding domain of ANT was pinpointed as the covalent binding site through ABPP, Peptide-centric Local Stability Assay (PELSA), and molecular docking. Functional assays using isolated gill mitochondria showed that 6PPDQ elicited an uncoupling-like mitochondrial respiratory response that was partially attenuated by the ANT inhibitor carboxyatractyloside (CATR), supporting the functional involvement of ANT in this gill-based model. Together, these findings nominate ANT as a candidate gill mitochondrial target associated with 6PPDQ-induced acute respiratory toxicity and demonstrate the utility of chemoproteomics for prioritizing mechanistically relevant protein interactions of emerging pollutants.

Animals↗
PubMed2026

Redox-activated chemistry for probing and perturbing the proteome: Lessons from protein redox switches.

Covalent drug discovery and chemical proteomics have historically relied on a nucleophilic logic, where electrophilic "warheads" react with nucleophilic amino acid side chains. While powerful, this paradigm probes only a single dimension of the protein's chemical surface. In contrast, biology leverages a second axis: redox potential. This is exemplified by the regulated redox proteome, where specific residues undergo reversible oxidation and reduction as functional post-translational modifications. Inspired by this natural machinery, researchers are developing redox-activated probes to label proteins at oxidizable residues and deploying similar chemistry to selectively perturb protein function. This review highlights recent advances in redox-activated covalent chemistry and explores its burgeoning potential for the development of next-generation targeted therapeutics.

Oxidation-Reduction↗
PubMed2026

Analysis of Confounding Factors in Reactive Cysteine Profiling Reveals Enhanced Chromatin-Protein Association via CDK7 Inhibition by THZ1.

Recent advances in activity-based proteome profiling (ABPP) have enabled the global mapping of cysteine ligandability, uncovering novel biological insights and opportunities for identifying disease vulnerabilities. While both live-cell-based and native-lysate-based ABPP have been applied, how cysteine ligandability differs between these systems and what factors influence these measurements remain unclear. Building on our previous development of a high-throughput TMT-ABPP workflow for native lysates, here we adapt the protocol for live cells and systematically compare cysteine ligandability across both platforms. Our analysis reveals three major contributors to the discrepancies: in-cellular cysteine accessibility, protein abundance changes, and protein relocalization. Notably, we highlight that the CDK7 inhibitor THZ1 induces substantial protein relocalization and promotes chromatin binding. Together, these results provide a practical framework for ABPP experimental design and data interpretation, supporting the more accurate application of ABPP in functional proteomics and drug discovery.

Cysteine↗
PubMed1988

Slow clearance of acylated, hybrid thrombolytic enzymes.

Two hybrid plasminogen activators, plasmin A-chain/t-PA B-chain and plasmin A-chain/u-PA B-chain have been synthesized and purified in sufficient yield to permit measurement of clearance in small laboratory animals. Each hybrid enzyme was reversibly acylated at the active centre to allow the pharmacokinetic profile to be followed using an activity-based method without interference from plasma inhibitors. The acylated plasmin/u-PA hybrid had a clearance half-life (t1/2) in guinea pigs of approximately 80 min, whereas acyl u-PA had a t1/2 of 3 min. The pharmacokinetic profile of the acylated plasmin/t-PA hybrid was measured in guinea pigs, rats and rabbits; the half-lives in all three species were 60-80 min compared to half-lives of acylated, native t-PA that were in the range 0.5-1.0 min. Thus, plasmin A-chain-containing, acylated hybrid enzymes are cleared some 30- to 100-fold more slowly than the acylated parent activators.

Acylation↗