RESUMEN
A saturation strategy focused on improving the selectivity and physicochemical properties of ATR inhibitor HTS hit 1 led to a novel series of highly potent and selective tetrahydropyrazolo[1,5-a]pyrazines. Use of PI3Kα mutants as ATR crystal structure surrogates was instrumental in providing cocrystal structures to guide the medicinal chemistry designs. Detailed DMPK studies involving cyanide and GSH as trapping agents during microsomal incubations, in addition to deuterium-labeled compounds as mechanistic probes uncovered the molecular basis for the observed CYP3A4 TDI in the series.
RESUMEN
Compound 13 was discovered through morphing of the ATR biochemical HTS hit 1. The ABI series was potent and selective for ATR. Incorporation of a 6-azaindole afforded a marked increase in cellular potency but was associated with poor PK and hERG ion channel inhibition. DMPK experiments established that CYP P450 and AO metabolism in conjunction with Pgp and BCRP efflux were major causative mechanisms for the observed PK. The series also harbored the CYP3A4 TDI liability driven by the presence of both a morpholine and an indole moiety. Incorporation of an adjacent fluorine or nitrogen into the 6-azaindole addressed many of the various medicinal chemistry issues encountered.
RESUMEN
UNLABELLED: Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost. IMPLICATIONS: These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Histonas/biosíntesis , Linfoma de Células del Manto/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Línea Celular Tumoral , Cromonas/administración & dosificación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Morfolinas/administración & dosificación , Mutación , ARN Interferente Pequeño , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.
Asunto(s)
Replicación del ADN , Histonas/metabolismo , Proteínas Quinasas/metabolismo , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Desoxirribonucleótidos/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/metabolismo , Ribonucleósido Difosfato Reductasa/genética , Ribonucleótidos/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.
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Desoxicitidina/análogos & derivados , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ribonucleósido Difosfato Reductasa , Relación Estructura-Actividad , GemcitabinaRESUMEN
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Óxidos N-Cíclicos , Quinasas Ciclina-Dependientes/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Histonas/metabolismo , Humanos , Immunoblotting , Indolizinas , Ratones , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/química , Compuestos de Piridinio/administración & dosificación , Compuestos de Piridinio/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/química , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes. Genomic dUMP is recognized by the base excision repair (BER) pathway. Using a synthetic lethal siRNA-screening approach, we systematically screened for components of BER that, when ablated, enhanced methotrexate response in a high content γ-H2A.X bioassay. We observed specific ablation of the mixed function DNA glycosylase/lyase Neil1 phenotypically enhanced several inhibitors of thymidine biosynthesis, as well as cellular phenotypes downstream of gemcitabine, cytarabine and clofarabine exposure. These synthetic lethal interactions were associated with significantly enhanced accumulation of γ-H2A.X and improved growth inhibition. Significantly, following TS pathway inhibition, addition of exogenous dTTP complemented the primary Neil1 γ-H2A.X phenotypes. Similarly, co-depletion of Neil1 with Cdc45, Cdc6, Cdc7 or DNA polymerase ß (PolB) suppressed Neil1 phenotypes. Conversely, co-depletion of Neil1 with the Rad17, Rad9 ATR, ATM and DNA-PK checkpoint/sensor proteins appears primarily epistatic to Neil1. These data suggest Neil1 may be a critical mediator of BER of incorporated dUMP following TS pathway inhibition.
Asunto(s)
ADN Glicosilasas/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Biomarcadores/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN Glicosilasas/genética , Reparación del ADN , Inhibidores Enzimáticos/metabolismo , Antagonistas del Ácido Fólico/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metotrexato/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Fenotipo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Quinazolinas/metabolismo , Tiofenos/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polalpha and not Polepsilon or Poldelta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polalpha and Chk1 causes an accumulation of gamma-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polalpha with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polalpha can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polalpha-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polalpha complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polalpha as an important component of the signal transduction cascade that activates the intra-S checkpoint.
Asunto(s)
ADN Polimerasa I/metabolismo , Replicación del ADN , Isoenzimas/metabolismo , Proteínas Quinasas/metabolismo , Antimetabolitos/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , ADN Polimerasa I/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Isoenzimas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Expression of the HIV-1 vpr gene in human and fission yeast cells displays multiple highly conserved activities, which include induction of cell cycle G2 arrest and cell death. We have previously characterized a yeast heat shock protein 16 (Hsp16) that suppresses the Vpr activities when it is overproduced in fission yeast. Similar suppressive effects were observed when the fission yeast hsp16 gene was overexpressed in human cells or in the context of viral infection. In this study, we further characterized molecular actions underlying the suppressive effect of Hsp16 on the Vpr activities. RESULTS: We show that the suppressive effect of Hsp16 on Vpr-dependent viral replication in proliferating T-lymphocytes is mediated through its C-terminal end. In addition, we show that Hsp16 inhibits viral infection in macrophages in a dose-dependent manner. Mechanistically, Hsp16 suppresses Vpr activities in a way that resembles the cellular heat shock response. In particular, Hsp16 activation is mediated by a heat shock factor (Hsf)-dependent mechanism. Interestingly, vpr gene expression elicits a moderate increase of endogenous Hsp16 but prevents its elevation when cells are grown under heat shock conditions that normally stimulate Hsp16 production. Similar responsive to Vpr elevation of Hsp and counteraction of this elevation by Vpr were also observed in our parallel mammalian studies. Since Hsf-mediated elevation of small Hsps occurs in all eukaryotes, this finding suggests that the anti-Vpr activity of Hsps is a conserved feature of these proteins. CONCLUSION: These data suggest that fission yeast could be used as a model to further delineate the potential dynamic and antagonistic interactions between HIV-1 Vpr and cellular heat shock responses involving Hsps.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Productos del Gen vpr/antagonistas & inhibidores , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Factores de Transcripción/metabolismo , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Proteínas de Unión al ADN/farmacología , Productos del Gen vpr/metabolismo , Genes prv , VIH-1/efectos de los fármacos , VIH-1/metabolismo , VIH-1/fisiología , Proteínas de Choque Térmico/farmacología , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Macrófagos/virología , Proteínas de Saccharomyces cerevisiae/farmacología , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Transcripción/farmacología , Replicación Viral/efectos de los fármacos , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Rad4(TopBP1) is a scaffold in a protein complex containing both replication proteins and checkpoint proteins and plays essential roles in both replication and checkpoint responses. We have previously identified four novel fission yeast mutants of rad4+(TopBP1) to explore how Rad4(TopBP1), a single protein, can play multiple roles in genomic integrity maintenance. Among the four novel mutants, rad4-c17(TopBP1) is a thermosensitive mutant. Here, we characterized rad4-c17(TopBP1) and identified a rad4-c17(TopBP1) allele specific suppressor named srr2+ (suppressor of Rad4(TopBP1) R2 domain). srr2+ has previously been identified as an environmental stress-responsive gene (GenBank accession number AL049644.1, locus spcc191.01). srr2+ null cells are sensitive to hydroxyurea (HU) at elevated temperatures. Deletion of srr2+ in rad4-c17(TopBP1) exacerbates the HU sensitivity of the mutant. Overexpression of srr2+ suppresses the rad4-c17(TopBP1) mutant sensitivity to temperature and HU and restores the compromised ability of rad4-c17(TopBP1) to activating Cds1 kinase in response to HU treatment. Furthermore, stress-activated MAPK, Spc1 (also known as StyI or Phh1), induces the expression and phosphorylation of the Srr2 protein. Significantly, environmental stress induces co-precipitation of Srr2 protein with Rad4(TopBP1), and the co-precipitation is compromised in the rad4-c17(TopBP1) mutant. These results have led us to propose a model; Rad4(TopBP1) exists in a large protein complex to coordinate genomic perturbations with checkpoint responses to maintain genomic integrity. In addition, when cells experience environmental stress, Rad4(TopBP1) associates with Srr2, an Spc1 MAPK-responsive protein, to survive the stress, potentially by providing a link of the Spc1 MAPK response to checkpoint responses.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/química , Proteínas de Choque Térmico/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/fisiología , Transglutaminasas/fisiología , Alelos , Secuencia de Aminoácidos , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/genética , Eliminación de Gen , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Temperatura , Transglutaminasas/genética , Rayos UltravioletaRESUMEN
Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.
Asunto(s)
Mutagénesis , Schizosaccharomyces/genética , Daño del ADN , Replicación del ADN , Recombinación GenéticaRESUMEN
Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.
Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transglutaminasas/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Activación Enzimática , Dosificación de Gen/genética , Mutación/genética , Fosforilación , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/citología , Técnicas del Sistema de Dos HíbridosRESUMEN
Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G(2) arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.
Asunto(s)
Productos del Gen vpr/efectos de los fármacos , Productos del Gen vpr/metabolismo , VIH-1/fisiología , Proteínas de Choque Térmico/farmacología , Proteínas de Schizosaccharomyces pombe , Replicación Viral/efectos de los fármacos , Muerte Celular , Línea Celular , Fase G2 , Productos del Gen vpr/genética , VIH-1/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Humanos , Schizosaccharomyces/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival. Collectively these changes are referred to as stationary phase. We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase. Ish1 is a nuclear envelope protein that is present throughout the cell cycle and whose expression is increased in response to stresses such as glucose and nitrogen starvation, as well as osmotic stress. Expression of Ish1 is regulated by the Spc1 MAPK pathway through the Atf1 transcription factor. Although overexpression of Ish1 is lethal, cells lacking ish1 exhibit reduced viability in stationary phase. Bis1 is a novel interacting partner of Ish1. Bis1 is the Schizosaccharomyces pombe member of the ES2 nuclear protein family found in Mus musculus, Drosophila melanogaster, Homo sapiens, and Arabidopsis thaliana. Overexpression of Bis1 results in a cell elongation phenotype, whereas bis1(-) cells exhibit a reduced viability in stationary phase similar to that seen in ish1(-) cells.