RESUMEN
Checkpoint kinases 1 and 2 (CHK1 and CHK2) are enzymes that are involved in the control of DNA damage. At the present time, these enzymes are some of the most important targets in the fight against cancer since their inhibition produces cytotoxic effects in carcinogenic cells. This paper proposes the use of spirostans (Sp), natural compounds, as possible inhibitors of the enzymes CHK1 and CHK2 from an in silico analysis of a database of 155 molecules (S5). Bioinformatics studies of molecular docking were able to discriminate between 13 possible CHK1 inhibitors, 13 CHK2 inhibitors and 1 dual inhibitor for both enzymes. The administration, distribution, metabolism, excretion and toxicity (ADMETx) studies allowed a prediction of the distribution and metabolism of the potential inhibitors in the body, as well as determining the excretion routes and the appropriate administration route. The best inhibition candidates were discriminated by comparing the enzyme-substrate interactions from 2D diagrams and molecular docking. Specific inhibition candidates were obtained, in addition to studying the dual inhibitor candidate and observing their stability in dynamic molecular studies. In addition, Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) interactions were analyzed to study the stability of interactions between the selected enzymes and spirostans resulting in the predominant gaps from HOMOCHKs to LUMOSp (Highest Occupied Molecular Orbital of CHKs-Lowest Unoccupied Molecular Orbital of spirostan). In brief, this study presents the selection inhibitors of CHK1 and CHK2 as a potential treatment for cancer using a combination of molecular docking and dynamics, ADMETx predictons, and HOMO-LUMO calculation for selection.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa de Punto de Control 2/metabolismo , Quinasa de Punto de Control 2/química , Humanos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Simulación por Computador , Antineoplásicos/química , Antineoplásicos/farmacología , Simulación de Dinámica MolecularRESUMEN
Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.
Asunto(s)
Quinasa de Punto de Control 2 , Síndromes Neoplásicos Hereditarios , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Síndromes Neoplásicos Hereditarios/genética , Estudios Prospectivos , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Dominios Proteicos , Masculino , Femenino , Persona de Mediana EdadRESUMEN
Familial inheritance in non-medullary thyroid cancer (NMTC) is an area that has yet to be adequately explored. Despite evidence suggesting strong familial clustering of non-syndromic NMTC, known variants still account for a very small percentage of the genetic burden. In a recent whole genome sequencing (WGS) study of five families with several NMTCs, we shortlisted promising variants with the help of our in-house developed Familial Cancer Variant Prioritization Pipeline (FCVPPv2). Here, we report potentially disease-causing variants in checkpoint kinase 2 (CHEK2), Ewing sarcoma breakpoint region 1 (EWSR1) and T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) in one family. Performing WGS on three cases, one probable case and one healthy individual in a family with familial NMTC left us with 112254 variants with a minor allele frequency of less than 0.1%, which was reduced by pedigree-based filtering to 6368. Application of the pipeline led to the prioritization of seven coding and nine non-coding variants from this family. The variant identified in CHEK2, a known tumor suppressor gene involved in DNA damage-induced DNA repair, cell cycle arrest, and apoptosis, has been previously identified as a germline variant in breast and prostate cancer and has been functionally validated by Roeb et al. in a yeast-based assay to have an intermediate effect on protein function. We thus hypothesized that this family may harbor additional disease-causing variants in other functionally related genes. We evaluated two further variants in EWSR1 and TIAM1 with promising in silico results and reported interaction in the DNA-damage repair pathway. Hence, we propose a polygenic mode of inheritance in this family. As familial NMTC is considered to be more aggressive than its sporadic counterpart, it is important to identify such susceptibility genes and their associated pathways. In this way, the advancement of personalized medicine in NMTC patients can be fostered. We also wish to reopen the discussion on monogenic vs polygenic inheritance in NMTC and instigate further development in this area of research.
Asunto(s)
Quinasa de Punto de Control 2/genética , Predisposición Genética a la Enfermedad , Proteína EWS de Unión a ARN/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/genética , Cáncer Papilar Tiroideo/genética , Secuencia de Aminoácidos , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Femenino , Frecuencia de los Genes , Genoma Humano , Humanos , Italia , Masculino , Linaje , Proteína EWS de Unión a ARN/química , Proteína EWS de Unión a ARN/metabolismo , Alineación de Secuencia , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/química , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Cáncer Papilar Tiroideo/metabolismo , Secuenciación Completa del GenomaRESUMEN
Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.
Asunto(s)
Quinasa de Punto de Control 2/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Neoplasias/enzimología , Neoplasias/genética , Animales , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Humanos , Tasa de Mutación , Especificidad por SustratoRESUMEN
When the herpes simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called Promyelocytic leukemia protein nuclear bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) infected cell polypeptide 0 (ICP0) that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here, we identify a bona fide SUMO-interacting motif in ICP0 (SIM-like sequence [SLS] 4) that is essential and sufficient to target SUMOylated proteins in PML NBs such as the PML and Sp100. We shown that phosphorylation of SLS4 creates new salt bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates the Ataxia-telangiectasia-mutated kinase-Checkpoint kinase 2 (ATM-Chk2) pathway to regulate the cell cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications: ubiquitination, SUMOylation, and phosphorylation.
Asunto(s)
Quinasa de Punto de Control 2/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteínas Virales/metabolismo , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Células HEK293 , Humanos , Fosforilación , Conformación Proteica , Dominios Proteicos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralAsunto(s)
Quinasa de Punto de Control 2/metabolismo , Ácido Graso Sintasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa de Punto de Control 2/química , Activación Enzimática , Ácido Graso Sintasas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas de Schizosaccharomyces pombe/química , Relación Estructura-ActividadRESUMEN
With the advent of rapid sequencing technologies, making sense of all the genomic variations that we see among us has been a major challenge. A plethora of algorithms and methods exist that try to address genome interpretation through genotype-phenotype linkage analysis or evaluating the loss of function/stability mutations in protein. Critical Assessment of Genome Interpretation (CAGI) offers an exceptional platform to blind-test all such algorithms and methods to assess their true ability. We take advantage of this opportunity to explore the use of molecular dynamics simulation as a tool to assess alteration of phenotype, loss of protein function, interaction, and stability. The results show that coarse-grained dynamics based protein flexibility analysis on 34 CHEK2 and 1719 CALM1 single mutants perform reasonably well for class-based predictions for phenotype alteration and two-thirds of the predicted scores return a correlation coefficient of 0.6 or more. When all-atom dynamics is used to predict altered stability due to mutations for Frataxin protein (8 cases), the predictions are comparable to the state-of-the-art methods. The competitive performance of our straightforward approach to phenotype interpretation contrasts with heavily trained machine learning approaches, and open new avenues to rationally improve genome interpretation.
Asunto(s)
Calmodulina/química , Quinasa de Punto de Control 2/química , Proteínas de Unión a Hierro/química , Mutación , Algoritmos , Calmodulina/genética , Quinasa de Punto de Control 2/genética , Estudios de Asociación Genética , Humanos , Proteínas de Unión a Hierro/genética , Aprendizaje Automático , Simulación de Dinámica Molecular , Fenotipo , Estabilidad Proteica , FrataxinaRESUMEN
The serine/threonine kinase, CHK2 (checkpoint kinase 2), is a key mediator in DNA damage response and a tumor suppressor, which is implicated in promoting cell cycle arrest, apoptosis and DNA repair. Accumulating evidence suggests that these functions are primarily exerted through phosphorylation downstream factors such as p53 and BRCA1. Recent studies have shown that ubiquitination is an important mode of regulation of CHK2. However, it remains largely unclear whether deubiquitinases participate in regulation of CHK2. Here, we report that a deubiquitinase, USP39, is a new regulator of CHK2. Mechanistically, USP39 deubiquitinates and stabilizes CHK2, which in turn enhances CHK2 stability. Short hairpin RNA (shRNA) mediated knockdown of USP39 led to deregulate CHK2, which resulted in compromising the DNA damage-induced G2/M checkpoint, decreasing apoptosis, and conferring cancer cells resistance to chemotherapy drugs and radiation treatment. Collectively, we identify USP39 as a novel regulator of CHK2 in the DNA damage response.
Asunto(s)
Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Resistencia a Antineoplásicos , Neoplasias Pulmonares/metabolismo , Tolerancia a Radiación , Proteasas Ubiquitina-Específicas/metabolismo , Células A549 , Ciclo Celular , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Estabilidad Proteica , Ubiquitinación , Regulación hacia ArribaRESUMEN
The CHEK2 gene and its encoded protein Chk2 have a well-known role in cancers, especially those related to breast cancer mediated through the BRCA1 gene. Additionally Chk2 has a crucial role in DNA repair, apoptosis and the cell cycle, which is why classification of variants of uncertain significance (VUS) is an area highly sought for a better elucidation of the "genomic effect" that results. Because it can often take years before enough clinical data is accumulated, and the costly and expensive functional analysis for individual variants presents a significant hurdle, it is important to identify other tools to help aid in clarifying the impact of specific variants on a protein's function and eventually the patient's health outcome. Here we describe a newly identified CHEK2 variant and analyze with an integrated approach combining genomics (whole exome analysis), clinical study, radiographic imaging, and protein informatics to identify and predict the functional impact of the VUS on the protein's behavior and predicted impact on the related pathways. The observed and analyzed defects in the protein were consistent with the expected clinical effect. Here, we support the use of personalized protein modeling and informatics and further our goal of developing a large-scale protein deposition archive for all protein-level VUS.
Asunto(s)
Quinasa de Punto de Control 2/genética , Biología Computacional/métodos , Genómica , Imagenología Tridimensional , Adulto , Carcinogénesis/genética , Carcinogénesis/patología , Quinasa de Punto de Control 2/química , Femenino , Humanos , Masculino , Modelos Moleculares , Neoplasias/genética , Linaje , Factores de Riesgo , Electricidad EstáticaRESUMEN
The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160â»180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs.
Asunto(s)
Epítopos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anticuerpos/inmunología , Sitios de Unión , Proteínas de Ciclo Celular/química , Quinasa de Punto de Control 2/química , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.
Asunto(s)
Receptores ErbB/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteoma/análisis , 5'-Nucleotidasa/química , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Acrilamidas , Compuestos de Anilina , Animales , Catepsinas/química , Catepsinas/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Cisteína/química , Receptores ErbB/genética , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Hígado/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Piperazinas/química , Piperazinas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteómica , Rodaminas/química , Trasplante HeterólogoRESUMEN
The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , Daño del ADN , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Fosfotreonina/metabolismo , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Dispersión del Ángulo Pequeño , Serina/química , Treonina/química , Treonina/metabolismoRESUMEN
BACKGROUND: Breast cancer (BC) is the most common malignancy in women and has a major heritable component. The risks associated with most rare susceptibility variants are not well estimated. To better characterise the contribution of variants in ATM, CHEK2, PALB2 and XRCC2, we sequenced their coding regions in 13 087 BC cases and 5488 controls from East Anglia, UK. METHODS: Gene coding regions were enriched via PCR, sequenced, variant called and filtered for quality. ORs for BC risk were estimated separately for carriers of truncating variants and of rare missense variants, which were further subdivided by functional domain and pathogenicity as predicted by four in silico algorithms. RESULTS: Truncating variants in PALB2 (OR=4.69, 95% CI 2.27 to 9.68), ATM (OR=3.26; 95% CI 1.82 to 6.46) and CHEK2 (OR=3.11; 95% CI 2.15 to 4.69), but not XRCC2 (OR=0.94; 95% CI 0.26 to 4.19) were associated with increased BC risk. Truncating variants in ATM and CHEK2 were more strongly associated with risk of oestrogen receptor (ER)-positive than ER-negative disease, while those in PALB2 were associated with similar risks for both subtypes. There was also some evidence that missense variants in ATM, CHEK2 and PALB2 may contribute to BC risk, but larger studies are necessary to quantify the magnitude of this effect. CONCLUSIONS: Truncating variants in PALB2 are associated with a higher risk of BC than those in ATM or CHEK2. A substantial risk of BC due to truncating XRCC2 variants can be excluded.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Neoplasias de la Mama/genética , Quinasa de Punto de Control 2/genética , Proteínas de Unión al ADN/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteínas de la Ataxia Telangiectasia Mutada/química , Quinasa de Punto de Control 2/química , Proteínas de Unión al ADN/química , Proteína del Grupo de Complementación N de la Anemia de Fanconi/química , Femenino , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Análisis de Secuencia de ProteínaRESUMEN
The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas HSP90 de Choque Térmico/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Benzoquinonas/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Transformada , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Reparación del ADN/efectos de los fármacos , Eliminación de Gen , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Humanos , Lactamas Macrocíclicas/farmacología , Síndrome de Nijmegen/genética , Síndrome de Nijmegen/metabolismo , Síndrome de Nijmegen/patología , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación/efectos de los fármacos , Mutación Puntual , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Interferencia de ARN , Ubiquitinación/efectos de los fármacosRESUMEN
Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently available assays for Rad53 activation are based on either electrophoretic mobility shifts or semiquantitative in situ autophosphorylation activity on protein blots. Here, we describe direct quantitative measures to assess Rad53 activity using immunoprecipitation kinase assays and quantitative mass spectrometric analysis of Rad53 activation loop autophosphorylation states. Both assays employ a highly specific Rad53 antibody, and thus enable the analysis of the untagged endogenous protein under physiological conditions. The principles of these assays are readily transferable to other protein kinases for which immunoprecipitation-grade antibodies are available, and thus potentially applicable to a wide range of eukaryotic signaling pathways beyond yeast.
Asunto(s)
Proteínas de Ciclo Celular/química , Quinasa de Punto de Control 2/química , Pruebas de Enzimas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Anticuerpos/química , Proteínas de Ciclo Celular/aislamiento & purificación , Quinasa de Punto de Control 2/aislamiento & purificación , Cromatografía Liquida , Activación Enzimática , Inmunoprecipitación , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
Ionizing radiation is the more effective therapy to reduce tumor growth through damaging the DNA of cells. In response to DNA damage, cells activate the checkpoint kinases such as CHK2, which signal to initiate repair processes and cell-cycle arrest, until the damaged DNA is repaired. At present, the development of CHK2 inhibitors has provided an interesting strategy for the treatment of cancer by introducing new radiation modifier agents. CHK2 inhibitors can contribute for the improvement of cancer therapy through sensitizing cancerous cells and radioprotection of normal cells against ionizing radiation. This review describes and discusses the most recent inhibitors of CHK2 and presents an evaluation of chemical structures and biological activities. As well as their role in cell growth during exposure to ionizing radiation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Quinasa de Punto de Control 2/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/metabolismo , Reparación del ADN/efectos de los fármacos , Humanos , Modelos MolecularesRESUMEN
The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Cristalografía por Rayos X , Dimerización , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , UltracentrifugaciónRESUMEN
Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome.
Asunto(s)
Empalme Alternativo/genética , Proteínas de Ciclo Celular/química , Quinasa de Punto de Control 2/química , Cristalografía por Rayos X , Proteínas de Saccharomyces cerevisiae/química , Proteasas Ubiquitina-Específicas/química , Secuencia de Aminoácidos , Catálisis , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , Conformación Proteica , Precursores del ARN/química , Precursores del ARN/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Empalmosomas/química , Empalmosomas/genética , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Espectrometría de Masas/métodos , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alquilantes/farmacología , Dominio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Daño del ADN/fisiología , Activación Enzimática , Homeostasis , Organismos Modificados Genéticamente , Fosforilación , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Fase S/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP. We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubation more favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP.