RESUMEN
The interior of the cell abounds with reactive species that can accumulate as non-enzymatic covalent modifications (NECMs) on biological macromolecules. These adducts interfere with many cellular processes, for example, by altering proteins' surface topology, enzymatic activity, or interactomes. Here, we discuss dynamic NECMs on chromatin, which serves as the cellular blueprint. We first outline the chemistry of NECM formation and then focus on the recently identified effects of their accumulation on chromatin structure and transcriptional output. We next describe the known cellular regulatory mechanisms that prevent or reverse NECM formation. Finally, we discuss recently developed chemical biology platforms for probing and manipulating these NECMs in vitro and in vivo.
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Código de Histonas , Histonas , Cromatina , Histonas/metabolismoRESUMEN
Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe's utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.
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Azidas/metabolismo , Histonas/metabolismo , Ribosa/metabolismo , Azidas/química , Glicosilación , Histonas/química , Estructura Molecular , Ribosa/químicaRESUMEN
Methylglyoxal (MGO) is a reactive dicarbonyl metabolite that modifies histones in vivo and induces changes in chromatin structure and function. Here we report the synthesis and application of a chemical probe for investigating MGO-glycation. A two-step synthesis of a Cu-click compatible alkynyl oxoaldehyde probe (AlkMGO) via sequential Dess-Martin and Riley oxidations is presented. This synthesis elevates the accessibility and utility of an important tool for tracking, enriching, and studying MGO-glycation to aid in understanding its underlying biochemical functions.
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Histonas , Piruvaldehído , GlicosilaciónRESUMEN
Cells are continuously subjected to an array of reactive/toxic chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these species, which can alter protein structure, function, stability, and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer's disease, cancer, and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modification of their tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. Despite the vast knowledge detailing the relationship between histone modifications and gene regulation, NECMs on histones remain poorly explored. A major reason for this difference stems from the fact that, unlike their enzymatically installed counterparts, NECMs are difficult to both control and test in vivo. Here, we review advances in our understanding of the effect non-enzymatic covalent modifications (NECMs) have on the epigenetic landscape, cellular fate, and their implications in disease. Cumulatively, this illustrates how the epigenetic code is directly toxified by chemicals and detoxified by corresponding eraser enzymes.
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Epigénesis Genética , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Epigenómica , Código de Histonas , Histonas/genética , HumanosRESUMEN
Because of their long half-lives and highly nucleophilic tails, histones are particularly susceptible to accumulating nonenzymatic covalent modifications, such as glycation. The resulting modifications can have profound effects on cellular physiology due to the regulatory role histones play in all DNA-templated processes; however, the complexity of Maillard chemistry on proteins makes tracking and enriching for glycated proteins a challenging task. Here, we characterize glyoxal (GO) modifications on histones using quantitative proteomics and an aniline-derived GO-reactive probe. In addition, we leverage this chemistry to demonstrate that the glycation regulatory proteins DJ-1 and GLO1 reduce levels of histone GO adducts. Finally, we employ a two-round pull-down method to enrich histone H3 GO glycation and map these adducts to specific chromatin regions.
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Glioxal , Histonas , Cromatina , Glicosilación , Glioxal/química , Glioxal/metabolismo , Histonas/metabolismo , ProteómicaRESUMEN
Histone nonenzymatic covalent modifications (NECMs) have recently emerged as an understudied class of posttranslational modifications that regulate chromatin structure and function. These NECMs alter the surface topology of histone proteins, their interactions with DNA and chromatin regulators, as well as compete for modification sites with enzymatic posttranslational modifications. NECM formation depends on the chemical compatibility between a reactive molecule and its target site, in addition to their relative stoichiometries. Here we survey the chemical reactions and conditions that govern the addition of NECMs onto histones as a manual to guide the identification of new physiologically relevant chemical adducts. Characterizing NECMs on chromatin is critical to attain a comprehensive understanding of this new chapter of the so-called "histone code".
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Cromatina/química , ADN/química , Histonas/química , Acilación , Antioxidantes/química , Sitios de Unión , Epigénesis Genética , Depuradores de Radicales Libres/química , Código de Histonas , Humanos , Conformación Molecular , Polisacáridos/química , Unión Proteica , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno/químicaRESUMEN
ß-Strands are a fundamental component of protein structure, and these extended peptide regions serve as binding epitopes for numerous protein-protein complexes. However, synthetic mimics that capture the conformation of these epitopes and inhibit selected protein-protein interactions are rare. Here we describe covalent and noncovalent ß-hairpin mimics of an extended strand region mediating the Tcf4/ß-catenin interaction. Our efforts afford a rationally designed lead for an underexplored region of ß-catenin, which has been the subject of numerous ligand discovery campaigns.
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Péptidos Cíclicos/metabolismo , Unión Proteica/efectos de los fármacos , Factor de Transcripción 4/metabolismo , beta Catenina/metabolismo , Secuencia de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Péptidos Cíclicos/química , Conformación ProteicaRESUMEN
The oncoprotein and Parkinson's disease-associated enzyme DJ-1/PARK7 has emerged as a promiscuous deglycase that can remove methylglyoxal-induced glycation adducts from both proteins and nucleotides. However, dissecting its structural and enzymatic functions remains a challenge due to the lack of potent, specific, and pharmacokinetically stable inhibitors targeting its catalytic site (including Cys106). To evaluate potential drug-like leads against DJ-1, we leveraged its deglycase activity in an enzyme-coupled, fluorescence lactate-detection assay based on the recent understanding of its deglycation mechanism. In addition, we developed assays to directly evaluate DJ-1's esterase activity using both colorimetric and fluorescent substrates. The resulting optimized assay was used to evaluate a library of potential reversible and irreversible DJ-1 inhibitors. The deglycase activity-oriented screening strategy described herein establishes a new platform for the discovery of potential anti-cancer drugs.
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Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.
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Bencilaminas/farmacología , Naftiridinas/farmacología , Proteómica/métodos , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Linaje de la Célula , Reparación del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Subunidades de Proteína , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , TranscriptomaRESUMEN
Epigenetic modifications, including those on DNA and histones, have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways. In turn, metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications. Recently, non-enzymatic covalent modifications (NECMs) by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms. Here, we summarize these recent advances in the identification and characterization of NECMs on nucleic acids, histones, and transcription factors, providing an additional mechanistic link between metabolism and epigenetics.
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Epigénesis Genética , Histonas/metabolismo , Metabolismo/genética , Ácidos Nucleicos/genética , Ácidos Nucleicos/metabolismo , Factores de Transcripción/metabolismo , Animales , Histonas/química , Humanos , Ácidos Nucleicos/química , Factores de Transcripción/químicaRESUMEN
BACKGROUND: The number of patients in whom mental illness progresses to stages in which acute, and often forced treatment is warranted, is on the increase across Europe. As a consequence, more patients are involuntarily admitted to Psychiatric Intensive Care Units (PICU). From several studies and reports it has become evident that important dissimilarities exist between PICU's. The current study seeks to describe organisational as well as clinical and patient related factors across ten PICU's in and outside the Amsterdam region, adjusted for or stratified by level of urbanization. METHOD/DESIGN: This paper describes the design of the Amsterdam Studies of Acute Psychiatry II (ASAP-II). This study is a prospective observational cohort study comparing PICU's in and outside the Amsterdam region on various patient characteristics, treatment aspects and recovery related variables. Dissimilarities were measured by means of collecting standardized forms which were filled out in the framework of care as usual, by means of questionnaires filled out by mental health care professionals and by means of extracting data from patient files for every consecutive patient admitted at participating PICU's during a specific time period. Urbanization levels for every PICU were calculated conform procedures as proposed by the Dutch Central Bureau for Statistics (CBS). DISCUSSION: The current study may provide a deeper understanding of the differences between psychiatric intensive care units that can be used to promote best practice and benchmarking procedures, and thus improve the standard of care.
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Áreas de Influencia de Salud/estadística & datos numéricos , Control de Formularios y Registros/métodos , Hospitales Psiquiátricos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Psiquiatría/organización & administración , Población Urbana/clasificación , Enfermedad Aguda , Estudios de Cohortes , Recolección de Datos , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Hospitales Psiquiátricos/normas , Hospitales Psiquiátricos/estadística & datos numéricos , Humanos , Masculino , Trastornos Mentales/clasificación , Trastornos Mentales/prevención & control , Países Bajos , Admisión del Paciente/estadística & datos numéricos , Alta del Paciente/estadística & datos numéricos , Estudios Prospectivos , Escalas de Valoración Psiquiátrica , Investigación Cualitativa , Proyectos de Investigación , Factores Socioeconómicos , Encuestas y Cuestionarios , Población Urbana/estadística & datos numéricosRESUMEN
Elucidating the metabolic changes that accompany disease states via metabolomics analysis of tissues has become an important avenue of exploration in biomarker and therapeutic target discovery. Conventional harvesting techniques rely on post-euthanasia tissue harvest which introduces ischemic conditions and subsequent metabolome changes that may ultimately introduce artifacts into final analyses. In this chapter, we present protocols for low-ischemia time rapid kidney tissue harvest followed by metabolite extraction for metabolomics studies in rodents.
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Anestesia General/métodos , Eutanasia Animal/métodos , Microextracción en Fase Líquida/métodos , Metaboloma , Metabolómica/métodos , Analgésicos , Animales , Congelación , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Isoflurano , Ketamina , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/instrumentación , Ratones , Pentobarbital , Percloratos/química , Ratas , Factores de TiempoRESUMEN
Many cellular processes as well as their associated pathologies are regulated by protein post-translational modifications (PTMs). Understanding the precise roles of these adducts hinges on the development of methods to robustly and site-specifically manipulate proteins in their physiological environments. Recently, ultrafast intein protein trans-splicing (PTS) was harnessed to incorporate site-specific modifications on cellular chromatin in live cells. In this chapter, we present the protocols for the generation of synthetic modifications on native chromatin as well as highlight the capabilities of this methodology.