RESUMEN
Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteostasis , Ubiquitinación , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , AnimalesRESUMEN
Besides the fundamental components of the chromatin, DNA and octameric histone, the non-histone chromatin proteins and non-coding RNA play a critical role in the organization of functional chromatin domains. The non-histone chromatin proteins therefore regulate the transcriptional outcome in both physiological and pathophysiological state as well. They also help to maintain the epigenetic state of the genome indirectly. Several transcription factors and histone interacting factors also contribute in the maintenance of the epigenetic states, especially acetylation by the induction of autoacetylation ability of p300/CBP. Alterations of KAT activity have been found to be causally related to disease manifestation, and thus could be potential therapeutic target.
Asunto(s)
Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética , Enfermedades Genéticas Congénitas/genética , Acetilación , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/ultraestructura , Proteína p300 Asociada a E1A/genética , Humanos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , ARN no Traducido/genética , Factores de Transcripción/genéticaRESUMEN
Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number of nonhistone proteins, p53 being the most prominent one. Recruitment of p300 to p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggests that p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined domains, where the structured domains are interlinked with unstructured linker regions. A crystal structure of the central domain of p300 encompassing Bromo, RING, PHD, and HAT domains demonstrates a compact module, where the HAT active site stays occluded by the RING domain. However, although p300 has a significant role in mediating the transcriptional activity of p53, only a few structural details on the complex of these two full-length proteins are available. Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex. The three-dimensional cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT domains occurs upon complex formation, which is likely required for exposing HAT active site and subsequent acetyltransferase activity. Our observation correlates well with previous studies showing that the presence of Bromodomain is obligatory for effective acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism whereby p300, following binding with p53, gets activated.
Asunto(s)
Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Humanos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteína p53 Supresora de Tumor/químicaRESUMEN
Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismo , Acetilación , Regulación Alostérica , Proteína Quinasa CDC2/química , Dominio Catalítico , Ciclo Celular , Células HEK293 , Células HeLa , Humanos , Modelos MolecularesRESUMEN
The transcriptional co-activator p300 is essential for p53 transactivation, although its precise role remains unclear. We report that p53 activates the acetyltransferase activity of p300 through the enhancement of p300 autoacetylation. Autoacetylated p300 accumulates near the transcription start sites accompanied by a similar enrichment of activating histone marks near those sites. Abrogation of p53-p300 interaction by a site-directed peptide inhibitor abolished p300-mediated histone acetylation, suggesting a crucial role played by the activation in p53-mediated gene regulation. Gain-of-function mutant p53, known to impart aggressive proliferative properties in tumor cells, also activates p300 autoacetylation. The same peptide abolished many of the gain-of-function properties of mutant p53 as well. Reversal of gain-of-function properties of mutant p53 suggests that molecules targeting the p53-p300 interface may be good candidates for anti-tumor drugs.
RESUMEN
Nucleophosmin (NPM1) is a nucleolar protein that is frequently overexpressed in various types of solid tumors. NPM1 is involved in several cellular processes that might contribute significantly to the increased proliferation potential of cancers. Previous reports suggest that NPM1 expression is highly increased in response to mitogenic and oncogenic signals, the mechanisms of which have not been elucidated extensively. Using constructs incorporating different fragments of the NPM1 promoter upstream to a Luciferase reporter gene, we have identified the minimal promoter of NPM1 and candidate transcription factors regulating NPM1 promoter activity by luciferase reporter assays. We have validated the roles of a few candidate factors at the transcriptional and protein level by quantitative reverse transcriptase PCR, immunoblotting and immunohistochemistry, and explored the mechanism of regulation of NPM1 expression using immunoprecipitation and chromatin immunoprecipitation assays. We show here that the expression of NPM1 is regulated by transcription factor c-fos, a protein that is strongly activated by growth factor signals. In addition, mutant p53 (R175H) overexpression also enhances NPM1 expression possibly through c-myc and c-fos. Moreover, both c-fos and mutant p53 are overexpressed in oral tumor tissues that showed NPM1 overexpression. Collectively, our results suggest that c-fos and mutant p53 R175H positively regulate NPM1 expression, possibly in synergism, that might lead to oncogenic manifestation.
Asunto(s)
Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica , Genes fos , Neoplasias de la Boca/patología , Mutación , Proteínas Nucleares/genética , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular , Humanos , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Pronóstico , Regiones Promotoras Genéticas , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: p300 (KAT3B) lysine acetyltransferase activity is modulated under different physiological and pathological contexts through the induction of trans-autoacetylation. This phenomenon is mediated by several factors, mechanisms of which are not fully understood. METHODS: Through acetyltransferase assays using full-length, baculovirus-expressed KATs, the specificity of NPM1-mediated enhancement of p300 autoacetylation was tested. Chaperone assays and tryptophan fluorescence studies were performed to evaluate the NPM1-induced protein folding. The NPM1 oligomer-defective mutant characterization was done by glutaraldehyde-crosslinking. The small-molecule inhibitor of NPM1 oligomerization was used to confirm the absolute requirement of multimeric NPM1 in vivo. Immunohistochemistry analysis of oral cancer patient samples was done to uncover the pathophysiological significance of NPM1-induced p300 autoacetylation. RESULTS: We find that the histone chaperone NPM1 is a specific inducer of p300 autoacetylation. Distinct from its histone chaperone activity, NPM1 is a molecular chaperone of p300. The biophysical experiments suggest that there is a reversible binding between NPM1 and p300 which can modulate p300 acetyltransferase activity. Disruption of NPM1 oligomerization suggests that oligomeric NPM1 is essential for the induction of p300 autoacetylation. Significantly, we observe a concomitant hyper-autoacetylation of p300 with overexpression of NPM1 in oral cancer samples. CONCLUSION: NPM1 can specifically modulate p300 acetyltransferase activity through the enhancement of autoacetylation. The molecular chaperone activity and oligomerization of NPM1 play a pivotal role in this phenomenon. GENERAL SIGNIFICANCE: NPM1 is overexpressed in several solid cancers, the significance of which is unknown. Induction of p300 autoacetylation could be the cause of NPM1-mediated tumorigenicity.
Asunto(s)
Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Neoplasias de la Lengua/metabolismo , Acetilación , Humanos , Nucleofosmina , Unión Proteica , Conformación Proteica , Neoplasias de la Lengua/patología , Células Tumorales CultivadasRESUMEN
EP300 is a member of the EP300/CBP family of lysine acetyltransferases (KATs) with multiple roles in development and physiology. Loss of EP300/CBP activity in humans causes a very rare congenital disorder called Rubinstein Taybi Syndrome (RSTS). The zebrafish genome has two co-orthologs of lysine acetyltransferase EP300 (KAT3B) in zebrafish viz. ep300a and ep300b. Chemical inhibition of Ep300 with C646, a competitive inhibitor and morpholino-based genetic knockdown of ep300a and ep300b cause defects in embryonic development reminiscent of the human RSTS syndrome. Remarkably, overexpression of Ep300a KAT domain results in near complete rescue of the jaw development defects, a characteristic feature of RSTS in human suggesting the dispensability of the protein-interaction and DNA-binding domains for at least some developmental roles of Ep300. We also perform a chemical screen and identify two inhibitors of deacetylases, CHIC35 and HDACi III, that can partially rescue the RSTS-like phenotypes. Thus, modeling rare human genetic disorders in zebrafish allows for functional understanding of the genes involved and can also yield small molecule candidates towards therapeutic goals.
Asunto(s)
Modelos Animales de Enfermedad , Proteína p300 Asociada a E1A , Embrión no Mamífero/embriología , Desarrollo Embrionario/genética , Técnicas de Silenciamiento del Gen , Síndrome de Rubinstein-Taybi , Proteínas de Pez Cebra , Pez Cebra , Animales , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Humanos , Síndrome de Rubinstein-Taybi/embriología , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/patología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The 'language' of covalent histone modifications translates environmental and cellular cues into gene expression. This vast array of post-translational modifications on histones are more than just covalent moieties added onto a protein, as they also form a platform on which crucial cellular signals are relayed. The reversible lysine acetylation has emerged as an important post-translational modification of both histone and non-histone proteins, dictating numerous epigenetic programs within a cell. Thus, understanding the complex biology of lysine acetylation and its regulators is essential for the development of epigenetic therapeutics. In this review, we will attempt to address the complexities of lysine acetylation in the context of tumorigenesis, their role in cancer progression and emphasize on the modalities developed to target lysine acetyltransferases towards cancer treatment.
Asunto(s)
Lisina Acetiltransferasas/metabolismo , Lisina/metabolismo , Neoplasias/metabolismo , Acetilación , Animales , Antineoplásicos/farmacología , Carcinogénesis/metabolismo , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
S100A4, a member of a calcium-regulated protein family, is involved in various cellular signaling pathways. From many studies over the last decade or so, it has become clear that it is involved in tumor metastasis, probably playing a determinative role. However, except the phenothiazine group of drugs, no significant inhibitor of S100A4 has been reported. Even the phenothiazines are very weak inhibitors of S100A4 action. In this study, we report design and development of a conformationally constrained helical peptide modeled on the non-muscle myosin peptide that binds to S100A4. This conformationally constrained peptide binds to S100A4 with a dissociation constant in the nanomolar range. We also synthesized a peptide for experimental control that bears several alanine mutations in the peptide-protein interface. We demonstrate that the former peptide specifically inhibits motility of H1299 and MCF-7 cells in a wound-healing assay. Structures of several S100A4-ligand complexes suggest that it may be possible to develop a smaller peptide-small molecule conjugate having high affinity for S100A4. Peptide-drug conjugates of this kind may play an important role in developing drug leads against this antimetastasis target.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Proteínas S100/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Células MCF-7 , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/farmacología , Miosina Tipo IIB no Muscular/química , Miosina Tipo IIB no Muscular/farmacología , Estructura Secundaria de Proteína , Proteína de Unión al Calcio S100A4 , Proteínas S100/metabolismoRESUMEN
There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC.
Asunto(s)
Curcumina/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Pirazoles/farmacología , Receptores Androgénicos/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Curcumina/farmacología , Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Células 3T3 NIH , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.
Asunto(s)
Proteína p300 Asociada a E1A/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Naftoquinonas/química , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular , Proteína p300 Asociada a E1A/química , Células HEK293 , Células HeLa , Humanos , Cinética , Lisina/química , Oxidación-Reducción , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/químicaRESUMEN
One of the important goals of most biological investigations is to classify and organize the experimental findings so that they are readily useful for deriving generalized rules. Although there is a huge amount of information on RNA structures in PDB, there are redundant files, ambiguous synthetic sequences etc. Moreover, a systematic hierarchical organization, reflecting RNA classification, is missing in PDB. In this investigation, we have classified all the available RNA structures from PDB through a programmatic approach. Hence, it would be now a simple assignment to regularly update the classification as and when new structures are released. The classification can further determine (i) a non-redundant set of RNA structures and (ii) if available, a set of structures of identical sequence and function, which can highlight structural polymorphism, ligand-induced conformational alterations etc. Presently, we have classified the available structures (2095 PDB entries having RNA chain longer than nine nucleotides solved by X-ray crystallography or NMR spectroscopy) into nine functional classes. The structures of same function and same source are mostly seen to be similar with subtle differences depending on their functional complexation. The web-server is available online at http://www.saha.ac.in/biop/www/HD-RNAS.html and is updated regularly.