RESUMEN
Recently, Pham et al. used an array of model systems to uncover a role for the enzyme methionine adenosyltransferase (MAT)-1A, which is mainly expressed in liver, in both sensing formaldehyde and regulating transcriptional responses that protect against it. This provides a new lens for understanding the effects of formaldehyde on gene regulation.
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Epigénesis Genética , Formaldehído , Metionina Adenosiltransferasa , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Humanos , Carbono/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genéticaRESUMEN
Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs-despite their direct relevance to covalent drug development-impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs. In a setup that contrasts nucleus- vs. cytoplasm-specific responsivity to reactive-metabolite modification (hydroxynonenylation), â¼40% of the top-enriched protein sensors investigated respond in compartments of nonprimary origin or where the canonical activity of the protein sensor is inoperative. CDK9-a primarily nuclear-localized kinase-was hydroxynonenylated only in the cytoplasm. Site-specific CDK9 hydroxynonenylation-which we identified in untreated cells-drives its nuclear translocation, downregulating RNA-polymerase-II activity, through a mechanism distinct from that of commonly used CDK9 inhibitors. Taken together, this work documents an unmet approach to quantitatively profile and decode localized and context-specific signaling/signal-propagation programs orchestrated by reactive covalent ligands.
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Proteínas/genética , Proteínas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Células RAW 264.7 , Transducción de Señal/fisiología , Transcripción Genética/genéticaRESUMEN
Borrowing some quotes from Harper Lee's novel "To Kill A Mockingbird" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.
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Biotina , Proteoma , BiotinilaciónRESUMEN
In these days of information overload and high-throughput analysis, it is easy to lose focus on the study of individual proteins. It is our conjecture that such investigations are still crucially important and offer uniquely penetrative insights. We thus present a discussion of biophysical methods to allow readers to get to know their protein of interest better. Although this perspective is not written with the expert in mind, we hope that for interdisciplinary scientists, or researchers who do not routinely perform biophysical analyses, the content will be helpful and inspiring.
RESUMEN
Understanding the targets and signaling roles of reactive electrophilic species (RES) at a specific cellular space and time has long been hampered by the reliance of the field on the bulk administration of excess RES from outside of cells and/or animals. Uncontrolled bolus methods provide limited understanding of target engagement for these individual nonenzymatic RES-modification events. REX technologies [targetable reactive electrophiles and oxidants (T-REX) and its genome-wide variant (G-REX)] were developed as a gateway to address these limitations. These protocols offer a new ability to both profile kinetically privileged sensors (KPSs) of RES at a systems level (G-REX™ profiling) and monitor signaling responses at the sensor protein-of-interest (POI)-specific level (T-REX™ delivery) with high spatiotemporal resolution. REX technologies are compatible with several model systems and are built on a HaloTag-targetable small-molecule photocaged precursor to a native RES.
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Proteínas/metabolismo , Transducción de Señal , Animales , Regulación de la Expresión Génica , Humanos , Cinética , Oxidantes/metabolismo , Proteínas/genéticaRESUMEN
Revolutionary proteomic strategies have enabled rapid profiling of the cellular targets of electrophilic small molecules. However, precise means to directly interrogate how these individual electrophilic modifications at low occupancy functionally reshape signaling networks have until recently been largely limited. We highlight here new methods that transcend proteomic platforms to forge a quantitative link between protein target-selective engagement and downstream signaling. We focus on recent progress in the study of non-enzyme-assisted signaling mechanisms and crosstalk choreographed by native reactive electrophilic species (RES). Using this as a model, we offer a long-term vision of how these toolsets together with fundamental biochemical knowledge of precision electrophile signaling may be harnessed to assist covalent ligand-target matching and ultimately amend disease-specific signaling dysfunction.
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Electrones , Oxidantes/farmacología , Proteómica , Transducción de Señal/efectos de los fármacos , Animales , HumanosRESUMEN
Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to deoxyribonucleotides. This chapter primarily presents an overview of this multifaceted and complex enzyme, covering RNR's role in enzymology, biochemistry, medicinal chemistry, and cell biology. It further focuses on RNR from mammals, whose interesting and often conflicting roles in health and disease are coming more into focus. We present pitfalls that we think have not always been dealt with by researchers in each area and further seek to unite some of the field-specific observations surrounding this enzyme. Our work is thus not intended to cover any one topic in extreme detail, but rather give what we consider to be the necessary broad grounding to understand this critical enzyme holistically. Although this is an approach we have advocated in many different areas of scientific research, there is arguably no other single enzyme that embodies the need for such broad study than RNR. Thus, we submit that RNR itself is a paradigm of interdisciplinary research that is of interest from the perspective of the generalist and the specialist alike. We hope that the discussions herein will thus be helpful to not only those wanting to tackle RNR-specific problems, but also those working on similar interdisciplinary projects centering around other enzymes.
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Ribonucleótido Reductasas , Animales , Desoxirribonucleótidos , Mamíferos , Oxidorreductasas , Ribonucleótido Reductasas/química , RibonucleótidosRESUMEN
For several years, drugs with reactive electrophilic appendages have been developed. These units typically confer prolonged residence time of the drugs on their protein targets, and may assist targeting shallow binding sites and/or improving the drug-protein target spectrum. Studies on natural electrophilic molecules have indicated that, in many instances, natural electrophiles use similar mechanisms to alter signaling pathways. However, natural reactive species are also endowed with other important mechanisms to hone signaling properties that are uncommon in drug design. These include ability to be active at low occupancy and elevated inhibitor kinetics. Herein, we discuss how we have begun to harness these properties in inhibitor design.
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Diseño de Fármacos , Transducción de Señal , Cinética , Proteínas/metabolismoRESUMEN
Here we provide a personal account of innovation and design principles underpinning a method to interrogate precision electrophile signaling that has come to be known as "REX technologies". This Account is framed in the context of trying to improve methods of target mining and understanding of individual target-ligand engagement by a specific natural electrophile and the ramifications of this labeling event in cells and organisms. We start by explaining from a practical standpoint why gleaning such understanding is critical: we are constantly assailed by a battery of electrophilic molecules that exist as a consequence of diet, food preparation, ineluctable endogenous metabolic processes, and potentially disease. The resulting molecules, which are detectable in the body, appear to be able to modify function of specific proteins. Aside from potentially being biologically relevant in their own right, these labeling events are essentially identical to protein-covalent drug interactions. Thus, on what proteins and even in what ways a covalent drug will work can be understood through the eyes of natural electrophiles; extending this logic leads to the postulate that target identification of specific electrophiles can inform on drug design. However, when we entered this field, there was no way to interrogate how a specific labeling event impacted a specific protein in an unperturbed cell. Methods to evaluate stoichiometry of labeling, and even chemospecificity of a specific phenotype were limited. There were further no generally accepted ways to study electrophile signaling that did not hugely disturb physiology.We developed T-REX, a method to study single-protein-specific electrophile engagement, to interrogate how single-protein electrophile labeling shapes pathway flux. Using T-REX, we discovered that labeling of several proteins by a specific electrophile, even at low occupancy, leads to biologically relevant signaling outputs. Further experimentation using T-REX showed that in some instances, single-protein isoforms were electrophile responsive against other isoforms, such as Akt3. Selective electrophile-labeling of Akt3 elicited inhibition of Akt-pathway flux in cells and in zebrafish embryos. Using these data, we rationally designed a molecule to selectively target Akt3. This was a fusion of the naturally derived electrophile and an isoform-nonspecific, reversible Akt inhibitor in phase-II trials, MK-2206. The resulting molecule was a selective inhibitor of Akt3 and was shown to fare better than MK-2206 in breast cancer xenograft mouse models. Recently, we have also developed a means to screen electrophile sensors that is unbiased and uses a precise burst of electrophiles. Using this method, dubbed G-REX, in conjunction with T-REX, we discovered new DNA-damage response upregulation pathways orchestrated by simple natural electrophiles. We thus emphasize how deriving a quantitative understanding of electrophile signaling that is linked to thorough and precise mechanistic studies can open doors to numerous medicinally and biologically relevant insights, from gleaning better understanding of target engagement and target mining to rational design of targeted covalent medicines.
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Preparaciones Farmacéuticas/química , Proteínas Proto-Oncogénicas c-akt/química , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Portadores de Fármacos/química , Evaluación Preclínica de Medicamentos , Femenino , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Humanos , Ligandos , Ratones , Oxidantes/química , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Trasplante HeterólogoRESUMEN
Our bodies produce a host of electrophilic species that can label specific endogenous proteins in cells. The signaling roles of these molecules are under active debate. However, in our opinion, it is becoming increasingly likely that electrophiles can rewire cellular signaling processes at endogenous levels. Attention is turning more to understanding how nuanced electrophile signaling in cells is. In this Perspective, we describe recent work from our laboratory that has started to inform on different levels of context-specific regulation of proteins by electrophiles. We discuss the relevance of these data to the field and to the broader application of electrophile signaling to precision medicine development, beyond the traditional views of their pleiotropic cytotoxic roles.
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Proteínas , Transducción de Señal , Proteínas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Here we draw insights from the latest serendipitous findings made on the opposing roles of a proposed drug-target protein Keap1. We weigh up how natural reactive electrophiles and electrophilic small-molecule drugs in clinical use directly impinge on seemingly conflicting, yet both Keap1-electrophile-modification-dependent, cell-survival- vs. cell-death-promoting behaviors. In the process, we convey how understanding reactive chemical-signal regulation at the single-protein-specific level is an enabling necessity in deconstructing otherwise intricate reactive-small-molecule-responsive cellular pathways. We hope this opinion piece further spurs the broader interests of basic and pharmaceutical research communities toward better understanding of molecular mechanisms underpinning reactive small-molecule-regulated signaling subsystems.
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Factor 2 Relacionado con NF-E2 , Transducción de Señal , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
In this tutorial review, we compare and contrast the chemical mechanisms of electrophile/oxidant sensing, and the molecular mechanisms of signal propagation. We critically analyze biological systems in which these different pathways are believed to be manifest and what the data really mean. Finally, we discuss applications of this knowledge to disease treatment and drug development.
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Oxidantes , Gemelos Monocigóticos , Humanos , Transducción de SeñalRESUMEN
The pressing need for innovation in drug discovery is spurring the emergence of drugs that turn on protein function, as opposed to shutting activity down. Several pharmacophores usher protein target gain-of-function, for instance: PROTACs promote protein target degradation; other drug candidates have been reported to function through dominant-negative inhibition of their target enzyme. Such classes of molecules are typically active at low target occupancy and display numerous advantages relative to canonical inhibitors, whose function is intrinsically tied to achieving, or exceeding a threshold occupancy. However, our ability to generally tap into gain-of-function processes through small molecule interventions is overall in its infancy. Herein, I outline how chemical biology is poised to help us bring this powerful idea to fruition. I further outline means through which gain-of-function events can be identified and harnessed.
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Diseño de Fármacos , Descubrimiento de Drogas , Proteínas/metabolismo , Proteolisis , Bibliotecas de Moléculas Pequeñas/farmacología , Humanos , Proteínas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Since the origins of DNA-based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate-limiting role in de novo deoxynucleoside-triphosphate (dNTP) biosynthesis. Paradoxically, the large subunit, RNR-α, of this obligatory two-component complex in mammals plays a context-specific antiproliferative role. There is little explanation for this dichotomy. Here, we show that RNR-α has a previously unrecognized DNA-replication inhibition function, leading to growth retardation. This underappreciated biological activity functions in the nucleus, where RNR-α interacts with ZRANB3. This process suppresses ZRANB3's function in unstressed cells, which we show to promote DNA synthesis. This nonreductase function of RNR-α is promoted by RNR-α hexamerization-induced by a natural and synthetic nucleotide of dA/ClF/CLA/FLU-which elicits rapid RNR-α nuclear import. The newly discovered nuclear signaling axis is a primary defense against elevated or imbalanced dNTP pools that can exert mutagenic effects irrespective of the cell cycle.
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Núcleo Celular/metabolismo , ADN Helicasas/antagonistas & inhibidores , Mutación , Ribonucleótido Reductasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células COS , Ciclo Celular , Proliferación Celular , Chlorocebus aethiops , Citosol/metabolismo , ADN/análisis , Daño del ADN , Replicación del ADN , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Células K562 , Ratones , Mutagénesis , Células 3T3 NIH , Unión Proteica , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA-sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using a Nrf2 activity-reporter zebrafish strain, we document that this post-transcriptional control of Nrf2 activity is conserved at the whole-vertebrate level.-Poganik, J. R., Long, M. J. C., Disare, M. T., Liu, X., Chang, S.-H., Hla, T., Aye, Y. Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.
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Proteína 1 Similar a ELAV/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Procesamiento Postranscripcional del ARN , Animales , Células Cultivadas , Proteína 1 Similar a ELAV/genética , Células HEK293 , Humanos , Ratones , Unión Proteica , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez CebraRESUMEN
The concept of cell signaling in the context of nonenzyme-assisted protein modifications by reactive electrophilic and oxidative species, broadly known as redox signaling, is a uniquely complex topic that has been approached from numerous different and multidisciplinary angles. Our Review reflects on five aspects critical for understanding how nature harnesses these noncanonical post-translational modifications to coordinate distinct cellular activities: (1) specific players and their generation, (2) physicochemical properties, (3) mechanisms of action, (4) methods of interrogation, and (5) functional roles in health and disease. Emphasis is primarily placed on the latest progress in the field, but several aspects of classical work likely forgotten/lost are also recollected. For researchers with interests in getting into the field, our Review is anticipated to function as a primer. For the expert, we aim to stimulate thought and discussion about fundamentals of redox signaling mechanisms and nuances of specificity/selectivity and timing in this sophisticated yet fascinating arena at the crossroads of chemistry and biology.
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Aldehídos/metabolismo , Alquenos/metabolismo , Cetonas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Aldehídos/química , Alquenos/química , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Diabetes Mellitus/etiología , Diabetes Mellitus/fisiopatología , Humanos , Cetonas/química , Esclerosis Múltiple/etiología , Esclerosis Múltiple/fisiopatología , Neoplasias/etiología , Neoplasias/fisiopatología , Oxidación-Reducción , Procesamiento Proteico-Postraduccional/fisiología , Proteínas/química , Proteínas/metabolismo , Especies Reactivas de Oxígeno/químicaRESUMEN
Precision cell signaling activities of reactive electrophilic species (RES) are arguably among the most poorly-understood means to transmit biological messages. Latest research implicates native RES to be a chemically-distinct subset of endogenous redox signals that influence cell decision making through non-enzyme-assisted modifications of specific proteins. Yet, fundamental questions remain regarding the role of RES as bona fide second messengers. Here, we lay out three sets of criteria we feel need to be met for RES to be considered as true cellular signals that directly mediate information transfer by modifying "first-responding" sensor proteins. We critically assess the available evidence and define the extent to which each criterion has been fulfilled. Finally, we offer some ideas on the future trajectories of the electrophile signaling field taking inspiration from work that has been done to understand canonical signaling mediators. Also see the video abstract here: https://youtu.be/rG7o0clVP0c.
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Electroquímica/métodos , Humanos , Oxidación-Reducción , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Protein-protein interactions (PPIs) are an effective means to orchestrate intricate biological processes required to sustain life. Approximately 650 000 PPIs underlie the human interactome; thus underscoring its complexity and the manifold signaling outputs altered in response to changes in specific PPIs. This minireview illustrates the growing arsenal of PPI assemblies and offers insights into how these varied PPI regulatory modalities are relevant to customized drug discovery, with a focus on cancer. First, known and emerging PPIs and PPI-targeted drugs of both natural and synthetic origin are categorized. Building on these discussions, the merits of PPI-guided therapeutics over traditional drug design are discussed. Finally, a compare-and-contrast section for different PPI blockers, with gain-of-function PPI interventions, such as PROTACS, is provided.
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Descubrimiento de Drogas , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas/metabolismo , Animales , HumanosRESUMEN
Isozyme-specific post-translational regulation fine tunes signaling events. However, redundancy in sequence or activity renders links between isozyme-specific modifications and downstream functions uncertain. Methods to study this phenomenon are underdeveloped. Here we use a redox-targeting screen to reveal that Akt3 is a first-responding isozyme sensing native electrophilic lipids. Electrophile modification of Akt3 modulated downstream pathway responses in cells and Danio rerio (zebrafish) and markedly differed from Akt2-specific oxidative regulation. Digest MS sequencing identified Akt3 C119 as the privileged cysteine that senses 4-hydroxynonenal. A C119S Akt3 mutant was hypomorphic for all downstream phenotypes shown by wild-type Akt3. This study documents isozyme-specific and chemical redox signal-personalized physiological responses.
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Proteínas Proto-Oncogénicas c-akt/metabolismo , Células HEK293 , Humanos , Isoenzimas/metabolismo , Lípidos , Oxidación-Reducción , Fenotipo , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.