RESUMEN
SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.
Asunto(s)
Modelos Moleculares , Imitación Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Supervivencia Celular , Humanos , Hidroliasas/química , Hidroliasas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Proteínas Nucleares/genética , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , ARN de Hongos/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes.
Asunto(s)
Proteínas Portadoras/fisiología , ADN Helicasas/fisiología , Sustancias Macromoleculares/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/biosíntesis , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN Helicasas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Células HeLa , Humanos , Ratones , Modelos Biológicos , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
DNA repair pathways enable tumour cells to survive DNA damage induced by external agents such as therapeutic treatments. Signalling cascades involved in these pathways comprise the DNA-dependent protein kinase (DNA-PK), Ataxia-telangiectasia mutated (ATM), ATM and Rad3 related (ATR) and checkpoint kinases I and 2 (Chk1/Chk2), among others. ATM and ATR phosphorylate, respectively, Chk2 and Chk1, leading to activation of checkpoints. Chk2 acts as a signal distributor, dispersing checkpoint signal to downstream targets such as p53, Cdc25A, Cdc25C, BRCA1 and E2F1. A role of Chk2 as a candidate tumour suppressor has been suggested based on both mouse genetics and somatic tumour studies. We will discuss here the possible role of this kinase in human carcinogenesis and the possibility to use it as a target to increment DNA damage in cancer cells in response to DNA-damaging therapies.
Asunto(s)
Neoplasias/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Quinasa de Punto de Control 2 , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.
Asunto(s)
Proteínas de Ciclo Celular/genética , Daño del ADN , Disqueratosis Congénita/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Estrés Oxidativo , Animales , Línea Celular , Disqueratosis Congénita/metabolismo , Disqueratosis Congénita/patología , Disqueratosis Congénita/terapia , Terapia Genética , Heterocromatina/genética , Heterocromatina/patología , Humanos , Ratones , Péptidos/genética , Péptidos/uso terapéutico , Telómero/genética , Telómero/patología , TransfecciónRESUMEN
BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.
Asunto(s)
Proteínas de Ciclo Celular/genética , Disqueratosis Congénita/genética , Disqueratosis Congénita/terapia , Mutación Missense/fisiología , Proteínas Nucleares/genética , Estabilidad del ARN/genética , ARN/metabolismo , Telomerasa/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/fisiología , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Proteínas de Ciclo Celular/uso terapéutico , Células Cultivadas , Disqueratosis Congénita/metabolismo , Disqueratosis Congénita/patología , Activación Enzimática/genética , Terapia Genética , Células HeLa , Humanos , Transferasas Intramoleculares/química , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Proteínas Nucleares/uso terapéutico , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , ARN/química , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/fisiología , Telomerasa/química , Valina/genéticaRESUMEN
Telomeres from most eukaryotes are composed of repeats of guanine-rich sequences whose main function is to preserve the end of the chromosomes. Telomeres are synthesised by a reverse transcriptase enzyme, telomerase (TERT), which forms part of a ribonucleoprotein complex containing also a RNA template molecule (TERC) and dyskerin. Exhaustion of telomeres during cell divisions triggers a DNA damage response that induces a senescence phenotype. The DNA damage machinery plays an essential role in maintaining the integrity of the genome and also detecting telomere shortening. However in some syndromes that involved mutations either in the telomerase complex genes or those involved in maintaining DNA secondary structure, such as the recQ helicase WRN, a higher frequency in the development of different types of malignancies is observed. We here describe the biology of some of these diseases, together with the molecular modifications in the telomerase complex genes and the impact of these alterations on the development of particular types of cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias/genética , Neoplasias/metabolismo , Telomerasa/deficiencia , Animales , Daño del ADN , Disqueratosis Congénita/genética , Exodesoxirribonucleasas/metabolismo , Genómica , Humanos , Mutación , ARN/metabolismo , RecQ Helicasas/metabolismo , Telomerasa/metabolismo , Telómero/ultraestructura , Síndrome de Werner/genética , Helicasa del Síndrome de WernerRESUMEN
Treatment of non-small cell lung cancer (NSCLC) with cisplatin has a level of antitumor activity still modest. We have shown previously that MKP1/DUSP1 inhibits cisplatin-induced apoptosis in NSCLC cells and is overexpressed in tumors from most patients with stage I-II NSCLC. Here, using different NSCLC cell lines we found that MKP1 and NF-kappaB are differentially expressed. We studied whether targeting MKP1, NF-kappaB or both affects cisplatin-induced cell death. MKP1 is expressed in H460 and H727 cells. H727 and H1299 cells showed constitutive phosphorylation of Akt and increased NF-kappaB activity than did H460 cells. H460-MKP1-siRNA-expressing cells (but not H727-MKP1-siRNA or H1299-MKP1-siRNA cells) exhibit a marked increase in cisplatin response compared with parental cells. Treatment with the PI3K inhibitor LY294002 or the NF-kappaB inhibitor BAY11-7082 enhanced cisplatin antitumor activity in parental H1299 cells but only weakly affected responses of H727 and H460 cells. MKP1-siRNA expression enhanced the chemosensitization effect of LY294002 and BAY11-7082 on H727 and H460 cells. Additionally, NSCLC cell lines with higher NF-kappaB-constitutive activation were the most sensitive to PS-341 (Bortezomib), a non-specific NF-kappaB inhibitor. This finding suggests the proteasome as a suitable strategy in treating NSCLC tumors with high constitutive NF-kappaB activity. Altogether, these results showed that either an activated PI3K/Akt/NF-kappaB pathway and/or high MKP1 was linked to reduced sensitivity to cisplatin in NSCLC cells. Inhibition of NF-kappaB or PI3K potently enhanced cisplatin cytotoxicity in cells with endogenous or genetically induced low MKP1 levels. These findings support the potential improvement in cisplatin responses by co-targeting NF-kappaB or Akt and MKP1.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Cisplatino/farmacología , Fosfatasa 1 de Especificidad Dual/metabolismo , Neoplasias Pulmonares/patología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Antineoplásicos/farmacología , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Fosfatasa 1 de Especificidad Dual/genética , Humanos , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Morfolinas/farmacología , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazinas/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacologíaRESUMEN
Dyskerin gene is mutated in patients with X-linked dyskeratosis congenita (X-DC), which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2-expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT was constitutively activated in GSE24-2 cells in a c-myc expression-dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the nuclease hypersensitive element-III (NHEIII) site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from patients with X-DC and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease.