RESUMEN
Iron-sulfur (Fe-S) clusters are essential redox-active metallocofactors participating in electron transfer, radical chemistry, primary metabolism, and gene regulation. Successful trafficking and incorporation of Fe-S clusters into target proteins are critical to proper cellular function. While biophysical studies of isolated Fe-S proteins provide insight into the structure and function of these inorganic cofactors, few strategies currently exist to directly interrogate Fe-S cluster binding within a cellular environment. Here, we describe a chemoproteomic platform to report on Fe-S cluster incorporation and occupancy directly within a native proteome, enabling the characterization of Fe-S biogenesis pathways and the identification of undiscovered Fe-S proteins.
Asunto(s)
Proteínas Hierro-Azufre , Proteómica , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/química , Proteómica/métodos , Unión Proteica , Proteoma , Hierro/metabolismo , Azufre/metabolismo , Oxidación-ReducciónRESUMEN
Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry approach to analyze the contribution of GrxD to the Fe-S proteome. Our results demonstrate that (1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, (2) GrxD is required for cluster delivery to ErpA under iron limitation, (3) GrxD is functionally distinct from other Fe-S trafficking proteins, and (4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Glutarredoxinas , Proteínas Hierro-Azufre , Hierro , Escherichia coli/metabolismo , Escherichia coli/genética , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Hierro/metabolismo , Estrés Fisiológico , LiasasRESUMEN
Ironsulfur (Fe-S) clusters, inorganic cofactors composed of iron and sulfide, participate in numerous essential redox, non-redox, structural, and regulatory biological processes within the cell. Though structurally and functionally diverse, the list of all proteins in an organism capable of binding one or more Fe-S clusters is referred to as its Fe-S proteome. Importantly, the Fe-S proteome is highly dynamic, with continuous cluster synthesis and delivery by complex Fe-S cluster biogenesis pathways. This cluster delivery is balanced out by processes that can result in loss of Fe-S cluster binding, such as redox state changes, iron availability, and oxygen sensitivity. Despite continued expansion of the Fe-S protein catalogue, it remains a challenge to reliably identify novel Fe-S proteins. As such, high-throughput techniques that can report on native Fe-S cluster binding are required to both identify new Fe-S proteins, as well as characterize the in vivo dynamics of Fe-S cluster binding. Due to the recent rapid growth in mass spectrometry, proteomics, and chemical biology, there has been a host of techniques developed that are applicable to the study of native Fe-S proteins. This review will detail both the current understanding of the Fe-S proteome and Fe-S cluster biology as well as describing state-of-the-art proteomic strategies for the study of Fe-S clusters within the context of a native proteome.
Asunto(s)
Proteínas Hierro-Azufre , Proteoma , Proteómica , Proteómica/métodos , Proteoma/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Oxidación-Reducción , Espectrometría de Masas/métodos , HumanosRESUMEN
The natural product holomycin contains a unique cyclic ene-disulfide and exhibits broad-spectrum antimicrobial activities. Reduced holomycin chelates metal ions with a high affinity and disrupts metal homeostasis in the cell. To identify cellular metalloproteins inhibited by holomycin, reactive-cysteine profiling was performed using isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). This chemoproteomic analysis demonstrated that holomycin treatment increases the reactivity of metal-coordinating cysteine residues in several zinc-dependent and iron-sulfur cluster-dependent enzymes, including carbonic anhydrase II and fumarase A. We validated that holomycin inhibits fumarase A activity in bacterial cells and diminishes the presence of iron-sulfur clusters in fumarase A. Whole-proteome abundance analysis revealed that holomycin treatment induces zinc and iron starvation and cellular stress. This study suggests that holomycin inhibits bacterial growth by impairing the functions of multiple metalloenzymes and sets the stage for investigating the impact of metal-binding molecules on metalloproteomes by using chemoproteomics.
Asunto(s)
Antibacterianos , Metaloproteínas , Antibacterianos/farmacología , Metaloproteínas/química , Metaloproteínas/metabolismo , Cisteína , Metales/química , Zinc , Hierro , HomeostasisRESUMEN
Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors involved in redox chemistry, radical generation and gene regulation. Common methods to monitor Fe-S clusters include spectroscopic analysis of purified proteins and autoradiographic visualization of radiolabeled iron distribution in proteomes. Here, we report a chemoproteomic strategy that monitors changes in the reactivity of Fe-S cysteine ligands to inform on Fe-S cluster occupancy. We highlight the utility of this platform in Escherichia coli by (1) demonstrating global disruptions in Fe-S incorporation in cells cultured under iron-depleted conditions, (2) determining Fe-S client proteins reliant on five scaffold, carrier and chaperone proteins within the Isc Fe-S biogenesis pathway and (3) identifying two previously unannotated Fe-S proteins, TrhP and DppD. In summary, the chemoproteomic strategy described herein is a powerful tool that reports on Fe-S cluster incorporation directly within a native proteome, enabling the interrogation of Fe-S biogenesis pathways and the identification of previously uncharacterized Fe-S proteins.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Chaperonas Moleculares , Proteoma/metabolismo , ProteómicaRESUMEN
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here, we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles, we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecule's effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligand's cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.
Asunto(s)
Fumarato Hidratasa , Síndromes Neoplásicos Hereditarios , Humanos , Cisteína , Ligandos , Síndromes Neoplásicos Hereditarios/diagnóstico , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/metabolismo , Fumaratos , ARNt Metiltransferasas , ARN de TransferenciaRESUMEN
Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections. Reactive oxygen species can drive tumor formation, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation dysregulates intracellular legumain processing and decreases the activity of the enzyme in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between an infection-induced oxidation site and tumorigenesis while underscoring the importance of cysteine reactivity in tumor growth.
Asunto(s)
Cisteína Endopeptidasas , Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Transformación Celular Neoplásica/metabolismo , Cisteína/metabolismo , Cisteína Endopeptidasas/metabolismo , Humanos , Oxidación-Reducción , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
Selenoproteins comprise a small group of selenocysteine (Sec) containing proteins, often involved in redox homeostasis. While Sec is functionally similar to cysteine (Cys), with both acting as protein-centered nucleophiles, chemoproteomic strategies employing electrophilic probes have often failed to rigorously identify Sec residues, due to their relatively low abundance with respect to Cys across a proteome. To improve the enrichment and detection of selenoproteins, herein we describe a chemoproteomic strategy that relies on the unique properties of Sec as compared to Cys, such as reduced pKa and the unique isotopic distribution of selenium. Low pH electrophilic probe labeling of mouse proteomes reduces Cys reactivity, resulting in increased identification of most soluble selenoproteins. This quantitative chemoproteomic platform provides a method to reliably measure changes in selenoprotein abundance across growth conditions as well as quantify inhibition by selenoprotein specific inhibitors, such as Auranofin.
Asunto(s)
Selenio , Selenocisteína , Animales , Cisteína/química , Concentración de Iones de Hidrógeno , Ratones , Proteoma , Selenocisteína/química , Selenocisteína/metabolismo , Selenoproteínas/química , Selenoproteínas/metabolismoRESUMEN
PURPOSE: To evaluate the impact of FDA's 2013 zolpidem Drug Safety Communications (DSCs), which recommended lowering the initial dose to mitigate drowsiness, on national estimates of zolpidem users and zolpidem exposure cases. METHODS: We analyzed trend changes of national zolpidem users from the IQVIA Total Patient Tracker (TPT) and zolpidem exposure cases reported to the National Poison Data System (NPDS), 2009-2018. To control for time varying confounding, the adjusted trends were analyzed using simple and controlled interrupted time series (ITS). We also adjusted for seasonal changes. Three sedating antidepressants were used together as a control. RESULTS: The national estimates of high-dose zolpidem users in TPT decreased significantly in the month immediately post-DSC; the absolute level decrease was -12.51 (95% CI: -14.12, -10.89) per 10 000 U.S. population relative to sedating antidepressants. The trend continuously decreased post-DSC, resulting in a 59% overall decrease by the end of the study period. There was a larger decrease in high-dose zolpidem use in females than in males. There was a level decrease of zolpidem exposure cases in the NPDS immediately post-DSC, -0.37 absolute decline (95% CI, -0.53, -0.20) per 10 000 national zolpidem users; or -1.33 absolute decline (95% CI, -1.54, -1.13) per 1000 total NPDS exposure cases relative to sedating antidepressants. Similar patterns were observed for cases reporting drowsiness. The results from the single ITS and controlled ITS were similar. CONCLUSIONS: Zolpidem users and exposure cases decreased significantly post-DSC, suggesting practitioners and patients became aware of and responded to the zolpidem DSCs.
Asunto(s)
Comunicación , Preparaciones Farmacéuticas , Femenino , Humanos , Hipnóticos y Sedantes/efectos adversos , Análisis de Series de Tiempo Interrumpido , Masculino , Estados Unidos/epidemiología , United States Food and Drug Administration , ZolpidemRESUMEN
A 2001 U.S. Government Accountability Office (GAO) report indicated 8 of 10 drugs withdrawn from the U.S. market between 1997 and 2000 posed greater risk to women than men. We examined drugs withdrawn from the market for safety-related reasons from January 1, 2001, to January 1, 2018. To be included, drugs must be listed as discontinued on Drugs@FDA and either listed in the Federal Register or cited in literature as being withdrawn for safety-related reasons. Biologics, over-the-counter products, and medical devices were excluded. During the 17-year time span, 19 drugs were withdrawn from the market for safety-related reasons, fewer drugs per year compared to the 3-year period examined in the GAO report. Food and Drug Administration (FDA) has not recommended the market removal of any drug approved since 2005 due to the time from the start of the Q wave to the end of the T wave (QT) interval prolongation resulting in torsades de pointes (TdP) or other abnormal heart rhythms. Furthermore, no drugs approved after the implementation of FDA's 2009 guidance on drug-induced liver injury (DILI) have been withdrawn because of hepatoxicity. All, but one of the drugs discontinued from the market for safety-related reasons during the period examined were approved between 1957 and 2002. TdP and DILI are two relevant examples of drug-induced adverse events posing greater risk to women than men. FDA has made measurable progress incorporating consideration of sex and gender differences into drug trial development and FDA review of these data, supporting inclusion of women in clinical trials, providing a comprehensive drug safety review, and advancing postmarket surveillance and risk assessment, thus strengthening FDA's ability to protect public health.
Asunto(s)
Preparaciones Farmacéuticas , Femenino , Humanos , Masculino , Medicamentos sin Prescripción , Responsabilidad Social , Estados Unidos , United States Food and Drug AdministrationRESUMEN
An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.
Asunto(s)
Cisteína/metabolismo , Fumarato Hidratasa/metabolismo , Fumaratos/metabolismo , Neoplasias Renales/metabolismo , Leiomiomatosis/metabolismo , Síndromes Neoplásicos Hereditarios/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Uterinas/metabolismo , Línea Celular Tumoral , Cisteína/genética , Fumarato Hidratasa/genética , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Leiomiomatosis/genética , Leiomiomatosis/patología , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Oral, live-attenuated rotavirus vaccines suffer from impaired immunogenicity and efficacy in low-income countries. Increasing the inoculum of vaccine might improve vaccine response, but this approach has been inadequately explored in low-income countries. METHODS: We performed a double-blind, parallel group, randomized controlled trial from June 2017 through June 2018 in the urban Mirpur slum of Dhaka, Bangladesh to compare vaccine take (primary outcome) among healthy infants randomized to receive either the standard dose or double the standard dose of oral Rotarix (GlaxoSmithKline) vaccine at 6 and 10â¯weeks of life. Infants with congenital malformations, birth or enrollment weight <2000â¯gm, known immunocompromising condition, enrollment in another vaccine trial, or other household member enrolled in the study were excluded. Infants were randomized using random permuted blocks. Vaccine take was defined as detection of post-vaccination fecal vaccine shedding by real-time reverse transcription polymerase chain reaction with sequence confirmation or plasma rotavirus-specific immunoglobulin A (RV-IgA) seroconversion 4â¯weeks following the second dose. RESULTS: 220 infants were enrolled and randomized (110 per group). 97 standard-dose and 92 high-dose infants completed the study per-protocol. For the primary outcome, no significant difference was observed between groups: vaccine take occurred in 62 (67%) high-dose infants versus 69 (71%) standard-dose infants (RR 0.92, 95% CI 0.67-1.24). However, in post-hoc analysis, children with confirmed vaccine replication had significantly increased RV-IgA responses, independent of the intervention. No significant adverse events related to study participation were detected. CONCLUSIONS: Administration of double the standard dose of an oral, live-attenuated rotavirus vaccine (Rotarix) did not improve vaccine take among infants in urban Dhaka, Bangladesh. However, improved immunogenicity in children with vaccine replication irrespective of initial inoculum provides further evidence for the need to promote in-host replication and improved gut health to improve oral vaccine response in low-income settings. ClinicalTrials.gov: NCT02992197.
Asunto(s)
Inmunización Secundaria/métodos , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/administración & dosificación , Administración Oral , Bangladesh/epidemiología , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Humanos , Lactante , Masculino , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/inmunología , Vacunas contra Rotavirus/inmunología , Resultado del Tratamiento , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in Escherichia coli However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant Astragalus bisulcatus that is reported to reject Sec in vitro Sequence analysis revealed a rare His â Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into E. coli and Saccharomyces cerevisiae CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate in vitro, we observed a reduction in the frequency of Sec misincorporation at Cys codons in vivo We surmise that the His â Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Planta del Astrágalo/enzimología , Escherichia coli/química , Ácido Selenioso/toxicidad , Selenocisteína/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/metabolismo , Prueba de Complementación Genética , Hidrólisis , Ácido Selenioso/metabolismoRESUMEN
Unlike most other tissues, the colon epithelium is exposed to high levels of H2S derived from gut microbial metabolism. H2S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H2S exposure as they harbor an efficient mitochondrial H2S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H2S poisoning. H2S at concentrations ≤20 µm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H2S concentrations as low as 5 µm poisoned cells. Mitochondrial H2S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD+/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H2S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H2S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H2S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming.
Asunto(s)
Metabolismo Energético , Sulfuro de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Colon/citología , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Cisteína/química , Cisteína/metabolismo , Metabolismo Energético/efectos de los fármacos , Humanos , Sulfuro de Hidrógeno/química , Sulfuro de Hidrógeno/farmacología , NAD/química , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Quinona Reductasas/antagonistas & inhibidores , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Mitochondria are cellular sites of diverse redox biology, including ROS production, iron-sulfur biogenesis, and secondary metabolism, which all rely on proteogenic reactive cysteine residues. Mass spectrometry-based proteomic methods to monitor the reactivity and functionality of cysteine residues across complex proteomes have greatly expanded over the past decade. Here we describe a mitochondrial isolation procedure coupled with cysteine-reactive IA labeling that affords identification and characterization of functional mitochondrial cysteine residues that were heretofore inaccessible to whole-cell proteomic analysis.
Asunto(s)
Cisteína/química , Proteoma/genética , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Mitocondrias/química , Mitocondrias/genética , Proteoma/químicaRESUMEN
Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
Asunto(s)
Fumaratos/metabolismo , Leiomiomatosis/metabolismo , Síndromes Neoplásicos Hereditarios/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Uterinas/metabolismo , Línea Celular Tumoral , Cromatografía Liquida/métodos , Cisteína , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Leiomiomatosis/genética , Modelos Biológicos , Síndromes Neoplásicos Hereditarios/genética , Proteómica , Transducción de Señal , Neoplasias Cutáneas/genética , Espectrometría de Masas en Tándem/métodos , Neoplasias Uterinas/genéticaRESUMEN
Cysteine residues are concentrated at key functional sites within proteins, performing diverse roles in metal binding, catalysis, and redox chemistry. Chemoproteomic platforms to interrogate the reactive cysteinome have developed significantly over the past 10 years, resulting in a greater understanding of cysteine functionality, modification, and druggability. Recently, chemoproteomic methods to examine reactive cysteine residues from specific subcellular organelles have provided significantly improved proteome coverage and highlights the unique functionalities of cysteine residues mediated by cellular localization. Here, the diverse physicochemical properties of the mammalian subcellular organelles are explored in the context of their effects on cysteine reactivity. The unique functions of cysteine residues found in the mitochondria and endoplasmic reticulum are highlighted, together with an overview into chemoproteomic platforms employed to investigate cysteine reactivity in subcellular organelles.
Asunto(s)
Cisteína/metabolismo , Orgánulos/metabolismo , Proteínas/metabolismo , Animales , Cisteína/análisis , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Humanos , Mitocondrias/química , Mitocondrias/metabolismo , Orgánulos/química , Oxidación-Reducción , Proteínas/análisis , Proteómica/métodosRESUMEN
Selenium (Se), as an essential trace element, plays crucial roles in many organisms including humans. The biological functions of selenium are mainly mediated by selenoproteins, a unique class of selenium-containing proteins in which selenium is inserted in the form of selenocysteine. Due to their low abundance and uneven tissue distribution, detection of selenoproteins within proteomes is very challenging, and therefore functional studies of these proteins are limited. In this study, we developed a computational method, named as selenium-encoded isotopic signature targeted profiling (SESTAR), which utilizes the distinct natural isotopic distribution of selenium to assist detection of trace selenium-containing signals from shotgun-proteomic data. SESTAR can detect femtomole quantities of synthetic selenopeptides in a benchmark test and dramatically improved detection of native selenoproteins from tissue proteomes in a targeted profiling mode. By applying SESTAR to screen publicly available datasets from Human Proteome Map, we provide a comprehensive picture of selenoprotein distributions in human primary hematopoietic cells and tissues. We further demonstrated that SESTAR can aid chemical-proteomic strategies to identify additional selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis. We believe SESTAR not only serves as a powerful tool for global profiling of native selenoproteomes, but can also work seamlessly with chemical-proteomic profiling strategies to enhance identification of target proteins, post-translational modifications, or protein-protein interactions.
RESUMEN
Mammalian selenocysteine (Sec)-containing proteins, selenoproteins, are important to (patho)physiological processes, including redox homeostasis. Sec residues have been recalcitrant to mass spectrometry-based chemoproteomic methods that enrich for reactive cysteine (Cys) residues with electrophilic chemical probes, despite confirmed reactivity of Sec with these electrophiles. Highly abundant Cys peptides likely suppress low-abundant Sec peptides. By exploiting the decreased pKa of Sec relative to Cys, we have developed a chemoproteomic platform that relies on low pH (pH 5.75) electrophile labeling, reducing Cys reactivity and enhancing identification of Sec-containing peptides across mouse tissues and cell lines. The utility of this Sec-profiling platform is underscored by evaluation of the selectivity of auranofin, an inhibitor of the selenoprotein, thioredoxin reductase, against both reactive Cys- and Sec-containing proteins. Platform limitations pertain to the non-physiological low-pH conditions that could perturb protein structure and function. Future work necessitates the discovery of Sec-selective electrophiles that function at physiological pH.