Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
1.
EMBO J ; 41(22): e109711, 2022 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-35929179

RESUMEN

Several kinds of stress promote the formation of three-stranded RNA:DNA hybrids called R-loops. Insufficient clearance of these structures promotes genomic instability and DNA damage, which ultimately contribute to the establishment of cancer phenotypes. Paraspeckle assemblies participate in R-loop resolution and preserve genome stability, however, the main determinants of this mechanism are still unknown. This study finds that in Multiple Myeloma (MM), AATF/Che-1 (Che-1), an RNA-binding protein fundamental to transcription regulation, interacts with paraspeckles via the lncRNA NEAT1_2 (NEAT1) and directly localizes on R-loops. We systematically show that depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids. We provide evidence that such failure to resolve R-loops causes sustained activation of a systemic inflammatory response characterized by an interferon (IFN) gene expression signature. Furthermore, elevated levels of R-loops and of mRNA for paraspeckle genes in patient cells are linearly correlated with Multiple Myeloma progression. Moreover, increased interferon gene expression signature in patients is associated with markedly poor prognosis. Taken together, our study indicates that Che-1/NEAT1 cooperation prevents excessive inflammatory signaling in Multiple Myeloma by facilitating the clearance of R-loops. Further studies on different cancer types are needed to test if this mechanism is ubiquitously conserved and fundamental for cell homeostasis.


Asunto(s)
Mieloma Múltiple , ARN Largo no Codificante , Humanos , Estructuras R-Loop , Mieloma Múltiple/genética , Paraspeckles , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Interferones/genética , Proteínas Represoras/metabolismo , Proteínas Reguladoras de la Apoptosis/genética
2.
Hum Mol Genet ; 32(22): 3153-3165, 2023 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-37565816

RESUMEN

Mutations in genes encoding nuclear pore proteins (NUPs) lead to the development of steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS). However, the precise molecular mechanisms by which NUP dysfunction contributes to podocyte injury preceding FSGS remain unclear. The tightly regulated activity of Yes-associated protein (YAP) and WW-domain-containing transcription regulator 1 (TAZ), the transcriptional effectors of the Hippo pathway, is crucial for podocytes and the maintenance of the glomerular filter. In this study, we investigate the impact of NUPs on the regulation of YAP/TAZ nuclear import and activity in podocytes. In unbiased interactome studies using quantitative label-free mass spectrometry, we identify the FSGS disease gene products NUP107, NUP133, NUP205, and Exportin-5 (XPO5) as components of YAP and TAZ protein complexes in podocytes. Moreover, we demonstrate that NUP205 is essential for YAP/TAZ nuclear import. Consistently, both the nuclear interaction of YAP/TAZ with TEA domain transcription factor 1 and their transcriptional activity were dependent on NUP205 expression. Additionally, we elucidate a regulatory feedback mechanism whereby YAP activity is modulated in response to TAZ-mediated NUP205 expression. In conclusion, this study establishes a connection between the FSGS disease protein NUP205 and the activity of the transcriptional regulators and Hippo effectors YAP and TAZ and it proposes a potential pathological role of YAP/TAZ dysregulation in podocytes of patients with pathogenic NUP205 variants.


Asunto(s)
Glomeruloesclerosis Focal y Segmentaria , Proteínas de Complejo Poro Nuclear , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glomeruloesclerosis Focal y Segmentaria/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Carioferinas , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Fosfoproteínas/genética , ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP
3.
Neuropathol Appl Neurobiol ; 48(6): e12837, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35839783

RESUMEN

AIMS: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model. MATERIALS AND METHODS: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)-dependent MB-prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1-depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA-Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. RESULTS: We found that monoallelic Nbn deletion increases SmoA1-dependent MB incidence. In addition to a defective DDR, Nbn+/- GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH-driven tumour allograft. CONCLUSIONS: Our study indicates that Nbn is haploinsufficient for SHH-MB development whereas full NbnKO is epistatic on SHH-driven MB development, thus revealing a gene dosage-dependent effect of Nbn inactivation on SHH-MB development.


Asunto(s)
Proteínas de Ciclo Celular , Neoplasias Cerebelosas , Proteínas de Unión al ADN , Meduloblastoma , Animales , Proteínas de Ciclo Celular/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Proteínas de Unión al ADN/genética , Dosificación de Gen , Genes Esenciales , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones Transgénicos
4.
Nucleic Acids Res ; 48(11): 5891-5906, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32421830

RESUMEN

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , ADN Ribosómico/genética , Genes de ARNr/genética , ARN Polimerasa I/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patología , Daño del ADN , ADN Ribosómico/metabolismo , Homeostasis , Humanos , Fosforilación , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Ribosomas/metabolismo
5.
Calcif Tissue Int ; 107(6): 603-610, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32875378

RESUMEN

We compared the effects of a nitrogen-containing bisphosphonate (N-BP), zoledronic acid (ZA), and an anti-mouse RANKL antibody (anti-mRANKL Ab) on the bone tissue pathology of a transgenic mouse model of human fibrous dysplasia (FD). For comparison, we also reviewed the histological samples of a child with McCune-Albright syndrome (MAS) treated with Pamidronate for 3 years. EF1α-GsαR201C mice with FD-like lesions in the tail vertebrae were treated with either 0.2 mg/kg of ZA at day 0, 7, and 14 or with 300 µg/mouse of anti-mRANKL Ab at day 0 and 21. All mice were monitored by Faxitron and histological analysis was performed at day 42. ZA did not affect the progression of the radiographic phenotype in EF1α-GsαR201C mice. FD-like lesions in the ZA group showed the persistence of osteoclasts, easily detectable osteoclast apoptotic activity and numerous "giant osteoclasts". In contrast, in the anti-mRANKL Ab-treated mice, osteoclasts were markedly reduced/absent, the radiographic phenotype reverted and the FD-like lesions were extensively replaced by newly formed bone. Numerous "giant osteoclasts" were also detected in the samples of the child with MAS. This study supports the hypothesis that osteoclasts per se, independently of their resorptive activity, are essential for development and expansion of FD lesions.


Asunto(s)
Displasia Fibrosa Ósea/tratamiento farmacológico , Células Gigantes , Osteoclastos , Ácido Zoledrónico/uso terapéutico , Animales , Difosfonatos , Modelos Animales de Enfermedad , Displasia Fibrosa Ósea/patología , Ratones , Ratones Transgénicos
6.
J Am Soc Nephrol ; 30(4): 564-576, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30867249

RESUMEN

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.


Asunto(s)
Células Epiteliales/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Hipoxia de la Célula/fisiología , Cilios/metabolismo , Células HEK293 , Humanos , Ratones , Unión Proteica
7.
Am J Pathol ; 186(5): 1128-39, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27105734

RESUMEN

Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.


Asunto(s)
Mitocondrias/fisiología , Podocitos/fisiología , Proteínas Represoras/deficiencia , Animales , Proteínas de Caenorhabditis elegans , Células Cultivadas , Células HEK293 , Humanos , Uniones Intercelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/fisiología , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/fisiopatología , Membranas Mitocondriales/fisiología , Membranas Mitocondriales/ultraestructura , Podocitos/ultraestructura , Prohibitinas , Proteinuria/etiología , Proteinuria/fisiopatología , Tacto/fisiología
8.
BMC Med Genet ; 18(1): 53, 2017 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-28499369

RESUMEN

BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.


Asunto(s)
Carcinoma de Células Renales/genética , Genes Supresores de Tumor , Neoplasias Renales/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Empalme del ARN , Proteínas Supresoras de Tumor/genética , Carcinoma de Células Renales/diagnóstico por imagen , Humanos , Neoplasias Renales/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos X
9.
EMBO J ; 31(20): 3961-75, 2012 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-22909821

RESUMEN

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Daño del ADN/fisiología , Proteínas Represoras/fisiología , Proteína p53 Supresora de Tumor/fisiología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Puntos de Control del Ciclo Celular , Daño del ADN/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias Endometriales/genética , Femenino , Amplificación de Genes , Dosificación de Gen , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos , Cadenas Ligeras de Miosina/metabolismo , Neuroblastoma/genética , Neuroblastoma/mortalidad , Presión Osmótica , Fosforilación , Pronóstico , Procesamiento Proteico-Postraduccional , Proteínas Represoras/genética
10.
Cancers (Basel) ; 15(14)2023 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-37509263

RESUMEN

Hypomorphic mutations in MRN complex genes are frequently found in cancer, supporting their role as oncosuppressors. However, unlike canonical oncosuppressors, MRN proteins are often overexpressed in tumor tissues, where they actively work to counteract DSBs induced by both oncogene-dependent RS and radio-chemotherapy. Moreover, at the same time, MRN genes are also essential genes, since the constitutive KO of each component leads to embryonic lethality. Therefore, even though it is paradoxical, MRN genes may work as oncosuppressive, oncopromoting, and essential genes. In this review, we discussed how alterations in the MRN complex impact the physiopathology of cancer, in light of our recent discoveries on the gene-dosage-dependent effect of NBS1 in Medulloblastoma. These updates aim to understand whether MRN complex can be realistically used as a prognostic/predictive marker and/or as a therapeutic target for the treatment of cancer patients in the future.

11.
J Biol Chem ; 286(16): 14237-45, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21357692

RESUMEN

Nephronophthisis is the most common genetic cause of end-stage renal failure during childhood and adolescence. Genetic studies have identified disease-causing mutations in at least 11 different genes (NPHP1-11), but the function of the corresponding nephrocystin proteins remains poorly understood. The two evolutionarily conserved proteins nephrocystin-1 (NPHP1) and nephrocystin-4 (NPHP4) interact and localize to cilia in kidney, retina, and brain characterizing nephronophthisis and associated pathologies as result of a ciliopathy. Here we show that NPHP4, but not truncating patient mutations, negatively regulates tyrosine phosphorylation of NPHP1. NPHP4 counteracts Pyk2-mediated phosphorylation of three defined tyrosine residues of NPHP1 thereby controlling binding of NPHP1 to the trans-Golgi sorting protein PACS-1. Knockdown of NPHP4 resulted in an accumulation of NPHP1 in trans-Golgi vesicles of ciliated retinal epithelial cells. These data strongly suggest that NPHP4 acts upstream of NPHP1 in a common pathway and support the concept of a role for nephrocystin proteins in intracellular vesicular transport.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cilios/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas/fisiología , Tirosina/química , Línea Celular , Proteínas del Citoesqueleto , Aparato de Golgi/metabolismo , Humanos , Enfermedades Renales Quísticas/metabolismo , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Distribución Tisular
12.
Curr Opin Nephrol Hypertens ; 20(4): 400-8, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21537177

RESUMEN

PURPOSE OF REVIEW: The introduction of Caenorhabditis elegans by Sydney Brenner to study 'how genes might specify the complex structures found in higher organisms' revolutionized molecular and developmental biology and pioneered a new research area to study organ development and cellular differentiation with this model organism. Here, we review the role of the nematode in renal research and discuss future perspectives for its use in molecular nephrology. RECENT FINDINGS: Although C. elegans does not possess an excretory system comparable with the mammalian kidney, various studies have demonstrated the conserved functional role of kidney disease genes in C. elegans. The finding that cystic kidney diseases can be considered ciliopathies is based to a great extent on research studying their homologues in the nematode's ciliated neurons. Moreover, proteins of the kidney filtration barrier play important roles in both correct synapse formation, mechanosensation and signal transduction in the nematode. Intriguingly, the renal cell carcinoma disease gene product von-Hippel-Lindau protein was shown to regulate lifespan in the nematode. Last but not least, the worm's excretory system itself expresses genes involved in electrolyte and osmotic homeostasis and may serve as a valuable tool to study these processes on a molecular level. SUMMARY: C. elegans has proven to be an incredibly powerful tool in studying various aspects of renal function, development and disease and will certainly continue to do so in the future.


Asunto(s)
Caenorhabditis elegans/metabolismo , Riñón/metabolismo , Longevidad , Mecanotransducción Celular , Animales , Transporte Biológico , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Regulación de la Expresión Génica , Genotipo , Humanos , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Longevidad/genética , Mecanotransducción Celular/genética , Modelos Animales , Fenotipo , Canales Catiónicos TRPP/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
13.
Kidney Int Rep ; 6(5): 1368-1378, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-34013115

RESUMEN

INTRODUCTION: Disease-causing mutations in the protocadherin FAT1 have been recently described both in patients with a glomerulotubular nephropathy and in patients with a syndromic nephropathy. METHODS: We identified 4 patients with FAT1-associated disease, performed clinical and genetic characterization, and compared our findings to the previously published patients. Patient-derived primary urinary epithelial cells were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblotting to identify possible alterations in Hippo signaling. RESULTS: Here we expand the spectrum of FAT1-associated disease with the identification of novel FAT1 mutations in 4 patients from 3 families (homozygous truncating variants in 3, compound heterozygous missense variants in 1 patient). All patients show an ophthalmologic phenotype together with heterogeneous renal phenotypes ranging from normal renal function to early-onset end-stage kidney failure. Molecular analysis of primary urine-derived urinary renal epithelial cells revealed alterations in the Hippo signaling cascade with a decreased phosphorylation of both the core kinase MST and the downstream effector YAP. Consistently, we found a transcriptional upregulation of bona fide YAP target genes. CONCLUSION: A comprehensive review of the here identified patients and those previously published indicates a highly diverse phenotype in patients with missense mutations but a more uniform and better recognizable phenotype in the patients with truncating mutations. Altered Hippo signaling and de-repressed YAP activity might be novel contributing factors to the pathomechanism in FAT1-associated renal disease.

14.
Oncogene ; 40(43): 6143-6152, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34508175

RESUMEN

MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.


Asunto(s)
Neoplasias Cerebelosas/tratamiento farmacológico , Meduloblastoma/tratamiento farmacológico , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/tratamiento farmacológico , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Sinergismo Farmacológico , Femenino , Amplificación de Genes/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Mutación , Neuroblastoma/genética , Neuroblastoma/patología , Ftalazinas/farmacología , Piperazinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Mol Microbiol ; 71(4): 1055-69, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19170884

RESUMEN

Biofilm production is thought to be an important step in many enterococcal infections. In several Gram-positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non-polar deletion mutant of a putative glucosyltransferase designated biofilm-associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030 Delta bgsA was devoid of diglucosyl-diacylglycerol (DGlcDAG), while monoglucosyl-diacylglycerol was overrepresented. The cell walls of 12030 Delta bgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild-type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA-deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalis Delta bgsA to Caco-2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030 Delta bgsA was cleared more rapidly from the bloodstream than the wild-type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.


Asunto(s)
Bacteriemia/microbiología , Biopelículas , Enterococcus faecalis/patogenicidad , Glucolípidos/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Animales , Células CACO-2 , Pared Celular/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Humanos , Lipopolisacáridos/metabolismo , Ratones , Mutagénesis Insercional , Eliminación de Secuencia , Ácidos Teicoicos/metabolismo , Virulencia
16.
J Am Soc Nephrol ; 20(12): 2513-7, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19797165

RESUMEN

Many genes are responsible for the modulation of lifespan in model organisms. In addition to regulating adaptive biologic responses that control stress signaling and longevity, some of these genes participate in tumor formation. The mechanisms that determine longevity and link regulation of lifespan with tumorigenesis are poorly understood. Here, we show that the tumor suppressor von Hippel-Lindau (VHL), which has widely known roles in renal carcinogenesis and the formation of kidney cysts, controls longevity in Caenorhabditis elegans. Loss of vhl-1 significantly increased lifespan and resulted in accelerated basal signaling of the p38 mitogen-activated protein kinase PMK-3. Furthermore, the VHL-1 effect on the regulation of lifespan was independent of the insulin/IGF-1-like signaling pathway, suggesting a mechanism for stress resistance that controls both lifespan and tumorigenesis. These findings define VHL-1 as a player in longevity signaling and connect aging, regulation of lifespan, and stress responses with formation of renal cell carcinomas.


Asunto(s)
Longevidad/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Humanos , Riñón/fisiología , Longevidad/fisiología , Sistema de Señalización de MAP Quinasas , Interferencia de ARN , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/antagonistas & inhibidores , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología
17.
Front Oncol ; 10: 919, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32587828

RESUMEN

The DNA damage response (DDR) is a complex signaling network that is activated upon genotoxic stress. It determines cellular fate by either activating cell cycle arrest or initiating apoptosis and thereby ensures genomic stability. The Apoptosis Antagonizing Transcription Factor (AATF/Che-1), an RNA polymerase II-interacting transcription factor and known downstream target of major DDR kinases, affects DDR signaling by inhibiting p53-mediated transcription of pro-apoptotic genes and promoting cell cycle arrest through various pathways instead. Specifically, AATF was shown to inhibit p53 expression at the transcriptional level and repress its pro-apoptotic activity by direct binding to p53 protein and transactivation of anti-apoptotic genes. Solid and hematological tumors of various organs exploit this function by overexpressing AATF. Both copy number gains and high expression levels of AATF were associated with worse prognosis or relapse of malignant tumors. Recently, a number of studies have enabled insights into the molecular mechanisms by which AATF affects both DDR and proliferation. AATF was found to directly localize to sites of DNA damage upon laser ablation and interact with DNA repair proteins. In addition, depletion of AATF resulted in increased DNA damage and decrease of both proliferative activity and genotoxic tolerance. Interestingly, considering the role of ribosomal stress in the regulation of p53, more recent work established AATF as ribosomal RNA binding protein and enabled insights into its role as an important factor for rRNA processing and ribosome biogenesis. This Mini Review summarizes recent findings on AATF and its important role in the DDR, malignancy, and ribosome biogenesis.

18.
PLoS One ; 15(11): e0238612, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33137122

RESUMEN

BACKGROUND: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). RESULTS: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK. CONCLUSION: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Neumonía Viral/diagnóstico , ARN Viral/metabolismo , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/virología , Humanos , Nasofaringe/virología , Pandemias , Neumonía Viral/virología , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Sensibilidad y Especificidad
19.
Front Oncol ; 10: 560, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32457828

RESUMEN

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases.

20.
Int J Artif Organs ; 32(9): 611-20, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19856273

RESUMEN

PURPOSE: Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. METHODS: The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. RESULTS: The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and biofilm associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. CONCLUSIONS: EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.


Asunto(s)
Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Enterococcus faecalis/patogenicidad , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana , Proteínas Bacterianas/genética , Composición de Base , Biopelículas , Caenorhabditis elegans , Modelos Animales de Enfermedad , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Enterococcus faecalis/metabolismo , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Infecciones por Bacterias Grampositivas/microbiología , Células HeLa , Humanos , Macrófagos/microbiología , Datos de Secuencia Molecular , Mutación , Factores de Tiempo , Virulencia , Factores de Virulencia/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA