RESUMO
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.
Assuntos
Epigênese Genética , Formaldeído , Metionina Adenosiltransferase , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Humanos , Carbono/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genéticaRESUMO
Current methods have limited ability in directly quantifying the extent of glutathionylation on specific protein-cysteines. In this issue of Cell Chemical Biology, Ahn et al.1 report G-ICAT (glutathione-based isotope-coded affinity tag), aimed at addressing this limitation. G-ICAT identifies Cysteine-692 within p120-catenin-a member of cadherin complex essential for cell-cell-contact maintenance-where C692-specific glutathionylation promotes E-cadherin destabilization.
Assuntos
Caderinas , CisteínaRESUMO
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.
Assuntos
Proteínas/genética , Proteínas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/fisiologia , Transcrição Gênica/genéticaRESUMO
Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1-ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Fumarato de Dimetilo/farmacologia , Imunossupressores/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Embrião de Mamíferos , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Peixe-ZebraRESUMO
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.
Assuntos
Preparações Farmacêuticas/química , Proteínas Proto-Oncogênicas c-akt/química , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Portadores de Fármacos/química , Avaliação Pré-Clínica de Medicamentos , Feminino , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Ligantes , Camundongos , Oxidantes/química , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transplante HeterólogoRESUMO
Off-target effects continue to impede disease interventions, particularly when targeting a specific protein within a family of similar proteins, such as kinase isoforms that play tumor-subtype-specific roles in cancers. Exploiting the specific electrophilic-metabolite-sensing capability of Akt3, versus moderate or no sensing, respectively, by Akt2 and Akt1, we describe a first-in-class functionally Akt3-selective covalent inhibitor [MK-H(F)NE], wherein the electrophilic core is derived from the native reactive lipid metabolite HNE. Mechanistic profiling and pathway interrogations point to retention of the metabolite's structure-as opposed to implicit electrophilicity-as being essential for biasing isoform preference, which we found translates to tumor-subtype specificity against pten-null triple-negative breast cancers (TNBCs). MK-H(F)NE further enables novel downstream target identification specific to Akt3-function in disease. In TNBC xenografts, MK-H(F)NE fares better than reversible pan-Akt-inhibitors and does not show commonly observed side-effects associated with Akt1-inhibition. Inhibitors derived from native-metabolite sensing are thus an enabling plan-of-action for unmasking kinase-isoform-biased molecular targets and tumor-subtype-specific interventions.
RESUMO
It is now clear that some cysteines on some proteins are highly tuned to react with electrophiles. Based on numerous studies, it is also established that electrophile sensing underpins rewiring of several critical signaling processes. These electrophile-sensing proteins, or privileged first responders (PFRs), are likely critically relevant for drug design. However, identifying PFRs remains a challenging and unsolved problem, despite the development of several high-throughput methods to ID proteins that react with electrophiles. More importantly, we remain unable to rank how different PFRs identified under different conditions relate to one another, in terms of sensing or signaling capacity. Here we evaluate different methods to assay sensing functions of proteins and discuss these methods in the context of developing a "ranking scheme." Based on theoretical and experimental evidence, we propose that T-REX-the only targeted-electrophile delivery tool presently available-is a reliable method to rank PFRs. Finally, we address to what extent electrophile sensing and downstream signaling are correlated. Based on our current data, we observe that such behaviors are indeed correlated. It is our hope that through this manuscript researchers from various arms of the stress signaling fields will focus on developing a quantitative understanding of precision electrophile labeling.
Assuntos
Elementos de Resposta Antioxidante , Proteínas , Transdução de Sinais , Cisteína , Desenho de Fármacos , Oxirredução , Proteínas/genética , Proteínas/metabolismoRESUMO
The key mRNA-binding proteins HuR and AUF1 are reported stress sensors in mammals. Intrigued by recent reports of sensitivity of these proteins to the electrophilic lipid prostaglandin A2 and other redox signals, we here examined their sensing abilities to a prototypical redox-linked lipid-derived electrophile, 4-hydroxynonenal (HNE). Leveraging our T-REX electrophile delivery platform, we found that only HuR, and not AUF1, is a kinetically-privileged sensor of HNE in HEK293T cells, and sensing functions through a specific cysteine, C13. Cells depleted of HuR, upon treatment with HNE, manifest unique alterations in cell viability and Nrf2-transcription-factor-driven antioxidant response (AR), which our recent work shows is regulated by HuR at the Nrf2-mRNA level. Mutagenesis studies showed that C13-specific sensing alone is not sufficient to explain HuR-dependent stress responsivities, further highlighting a complex context-dependent layer of Nrf2/AR regulation through HuR.
RESUMO
Ribonucleotide reductase (RNR) is an essential enzyme in DNA biogenesis and a target of several chemotherapeutics. Here, we investigate how anti-leukemic drugs (e.g., clofarabine [ClF]) that target one of the two subunits of RNR, RNR-α, affect non-canonical RNR-α functions. We discovered that these clinically approved RNR-inhibiting dATP-analogs inhibit growth by also targeting ZRANB3-a recently identified DNA synthesis promoter and nuclear-localized interactor of RNR-α. Remarkably, in early time points following drug treatment, ZRANB3 targeting accounted for most of the drug-induced DNA synthesis suppression and multiple cell types featuring ZRANB3 knockout/knockdown were resistant to these drugs. In addition, ZRANB3 plays a major role in regulating tumor invasion and H-rasG12V-promoted transformation in a manner dependent on the recently discovered interactome of RNR-α involving select cytosolic-/nuclear-localized protein players. The H-rasG12V-promoted transformation-which we show requires ZRANB3-supported DNA synthesis-was efficiently suppressed by ClF. Such overlooked mechanisms of action of approved drugs and a previously unappreciated example of non-oncogene addiction, which is suppressed by RNR-α, may advance cancer interventions.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Clofarabina/farmacologia , DNA Helicases/antagonistas & inibidores , Ribonucleotídeo Redutases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA Helicases/deficiência , DNA Helicases/metabolismo , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/biossíntese , Células HeLa , Humanos , Ribonucleotídeo Redutases/metabolismoRESUMO
Significance: Electrophile signaling is coming into focus as a bona fide cell signaling mechanism. The electrophilic regulation occurs typically through a sensing event (i.e., labeling of a protein) and a signaling event (the labeling event having an effect of the proteins activity, association, etc.). Recent Advances: Herein, we focus on the first step of this process, electrophile sensing. Electrophile sensing is typically a deceptively simple reaction between the thiol of a protein cysteine, of which there are around 200,000 in the human proteome, and a Michael acceptor, of which there are numerous flavors, including enals and enones. Recent data overall paint a picture that despite being a simple chemical reaction, electrophile sensing is a discerning process, showing labeling preferences that are often not in line with reactivity of the electrophile. Critical Issues: With a view to trying to decide what brings about highly electrophile-reactive protein cysteines, and how reactive these sensors may be, we discuss aspects of the thermodynamics and kinetics of covalent/noncovalent binding. Data made available by several laboratories indicate that it is likely that specific proteins exhibit highly stereo- and chemoselective electrophile sensing, which we take as good evidence for recognition between the electrophile and the protein before forming a covalent bond. Future Directions: We propose experiments that could help us gain a better and more quantitative understanding of the mechanisms through which sensing comes about. We further extoll the importance of performing more detailed experiments on labeling and trying to standardize the way we assess protein-specific electrophile sensing.
Assuntos
Oxirredução , Transdução de Sinais , Cisteína/metabolismo , Humanos , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Stereotyped as a nexus of dNTP synthesis, the dual-subunit enzyme - ribonucleotide reductase (RNR) - is coming into view as a paradigm of oligomerization and moonlighting behavior. In the present issue of 'omics', we discuss what makes the larger subunit of this enzyme (RNR-α) so interesting, highlighting its emerging cellular interactome based on its unique oligomeric dynamism that dictates its compartment-specific occupations. Linking the history of the field with the multivariable nature of this exceedingly sophisticated enzyme, we further discuss implications of new data pertaining to DNA-damage response, S-phase checkpoints, and ultimately tumor suppression. We hereby hope to provide ideas for those interested in these fields and exemplify conceptual frameworks and tools that are useful to study RNR's broader roles in biology.
Assuntos
Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Animais , Dano ao DNA , DNA Helicases/química , DNA Helicases/metabolismo , Desoxiadenosinas/metabolismo , Humanos , Modelos Moleculares , Mapas de Interação de Proteínas , Estrutura Quaternária de ProteínaRESUMO
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.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Células Cultivadas , Proteína Semelhante a ELAV 1/genética , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
Ability to faithfully report drug-target interactions constitutes a major critical parameter in preclinical/clinical settings. Yet the assessment of target engagement remains challenging, particularly for promiscuous and/or polypharmacologic ligands. Drawing from our improved insights into native electrophile signaling and emerging technologies that profile and interrogate these non-enzyme-assisted signaling subsystems, we posit that 'trained' polypharmocologic covalent inhibitors can be designed. Accumulating evidence indicates that electrophile-modified states at fractional occupancy can alter cell fate. Thus, by understanding sensing preferences and ligandable regions favored by the natural electrophilic signals at individual protein-ligand resolution, we can better evaluate target engagement and develop a function-guided understanding of polypharmacology.
Assuntos
Produtos Biológicos/farmacologia , Preparações de Ação Retardada , Polifarmacologia , Humanos , Ligantes , Ligação Proteica , Transdução de SinaisRESUMO
A Cu(II)-catalyzed diastereoselective Michael/aldol cascade approach is used to accomplish concise total syntheses of cardiotonic steroids with varying degrees of oxygenation including cardenolides ouabagenin, sarmentologenin, 19-hydroxysarmentogenin, and 5- epi-panogenin. These syntheses enabled the subsequent structure activity relationship (SAR) studies on 37 synthetic and natural steroids to elucidate the effect of oxygenation, stereochemistry, C3-glycosylation, and C17-heterocyclic ring. Based on this parallel evaluation of synthetic and natural steroids and their derivatives, glycosylated steroids cannogenol-l-α-rhamnoside (79a), strophanthidol-l-α-rhamnoside (92), and digitoxigenin-l-α-rhamnoside (97) were identified as the most potent steroids demonstrating broad anticancer activity at 10-100 nM concentrations and selectivity (nontoxic at 3 µM against NIH-3T3, MEF, and developing fish embryos). Further analyses indicate that these molecules show a general mode of anticancer activity involving DNA-damage upregulation that subsequently induces apoptosis.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Ouabaína/análogos & derivados , Oxigênio/química , Animais , Antineoplásicos/química , Linhagem Celular , Técnicas de Química Sintética , Camundongos , Ouabaína/síntese química , Ouabaína/química , Ouabaína/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Descoberta de Drogas , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas/metabolismo , Animais , HumanosRESUMO
Challenging the paradigm of SECIS-dependent selenoprotein translation, in this issue of Cell Chemical BiologyGuo et al. (2018) introduce a new selenoprotein profiling platform with which they identify novel selenoproteins apparently lacking SECIS. With increased interest in covalent targeting of reactive Sec residues in drug discovery, their method adds a valuable contribution toward expanding the druggable human proteome.
Assuntos
Selenocisteína , Selenoproteínas , Humanos , Espectrometria de Massas , Proteoma , Proteínas Proto-Oncogênicas c-retRESUMO
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.
Assuntos
Aldeídos/metabolismo , Alcenos/metabolismo , Cetonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Aldeídos/química , Alcenos/química , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Diabetes Mellitus/etiologia , Diabetes Mellitus/fisiopatologia , Humanos , Cetonas/química , Esclerose Múltipla/etiologia , Esclerose Múltipla/fisiopatologia , Neoplasias/etiologia , Neoplasias/fisiopatologia , Oxirredução , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/química , Proteínas/metabolismo , Espécies Reativas de Oxigênio/químicaRESUMO
Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true kcat/Km conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.
RESUMO
Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the ß subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and ß2. Together, these structures support a model for RNR inhibition in which ß2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.
Assuntos
Multimerização Proteica , Ribonucleotídeo Redutases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Microscopia Crioeletrônica , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ribonucleotídeo Redutases/metabolismoRESUMO
Small heat shock protein (sHSP)-B7 (HSPB7) is a muscle-specific member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically privileged sensor that is able to react rapidly with native reactive electrophilic species (RES), when only substoichiometric amounts of RES are available in proximity to Hspb7 expressed in living cells. Among the two Hspb7-cysteines, this RES sensing is fulfilled by a single cysteine (C117). Purification and characterizations in vitro reveal that the rate for RES adduction is among the most efficient reported for protein-cysteines with native carbonyl-based RES. Covalent-ligand binding is accompanied by structural changes (increase in ß-sheet-content), based on circular dichroism analysis. Among the two cysteines, only C117 is conserved across vertebrates; we show that the human ortholog is also capable of RES sensing in cells. Furthermore, a cancer-relevant missense mutation reduces this RES-sensing property. This evolutionarily conserved cysteine-biosensor may play a redox-regulatory role in cardioprotection.