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1.
Cell ; 182(4): 1009-1026.e29, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32730809

RESUMO

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.


Assuntos
Cisteína/metabolismo , Ligantes , Linfócitos T/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Acrilamidas/química , Acrilamidas/farmacologia , Células Cultivadas , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Proteoma/química , Proteoma/metabolismo , Estereoisomerismo , Linfócitos T/citologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/metabolismo
2.
Mol Cell ; 83(10): 1725-1742.e12, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37084731

RESUMO

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.


Assuntos
Proteômica , Fatores de Transcrição , Humanos , Proteômica/métodos , Cisteína/metabolismo , Ligantes
3.
J Proteome Res ; 22(11): 3652-3659, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37819886

RESUMO

The growing complexity and volume of proteomics data necessitate the development of efficient software tools for peptide identification and quantification from mass spectra. Given their central role in proteomics, it is imperative that these tools are auditable and extensible─requirements that are best fulfilled by open-source and permissively licensed software. This work presents Sage, a high-performance, open-source, and freely available proteomics pipeline. Scalable and cloud-ready, Sage matches the performance of state-of-the-art software tools while running an order of magnitude faster.


Assuntos
Proteômica , Software , Peptídeos , Espectrometria de Massas , Bases de Dados de Proteínas , Algoritmos
4.
Nat Chem ; 13(11): 1081-1092, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34504315

RESUMO

Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small-molecule-lysine interactions captured by the entire library. We used these latter 'scout' electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein-RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.


Assuntos
Lisina/química , Proteoma/química , Células HEK293 , Humanos , Ligantes , Proteômica/métodos , Relação Estrutura-Atividade
5.
Nat Chem ; 9(12): 1181-1190, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29168484

RESUMO

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.


Assuntos
Lisina/química , Proteoma/química , Humanos , Ligantes , Proteômica
6.
Cell Chem Biol ; 24(11): 1388-1400.e7, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-28965727

RESUMO

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.


Assuntos
Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/química , Proteoma/análise , 5'-Nucleotidase/química , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Acrilamidas , Compostos de Anilina , Animais , Catepsinas/química , Catepsinas/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cisteína/química , Receptores ErbB/genética , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Piperazinas/química , Piperazinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteômica , Rodaminas/química , Transplante Heterólogo
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