RESUMO
BACKGROUND: C3 Glomerulopathy (C3G) is a rare glomerular disease caused by dysregulation of the complement pathway. Based on its pathophysiology, treatment with the monoclonal antibody eculizumab targeting complement C5 may be a therapeutic option. Due to the rarity of the disease, observational data on the clinical response to eculizumab treatment is scarce. METHODS: Fourteen patients (8 female, 57%) treated for C3 glomerulopathy at the medical center of the University of Freiburg between 2013 and 2022 were included. Subjects underwent biopsy before enrollment. Histopathology, clinical data, and response to eculizumab treatment were analyzed. Key parameters to determine the primary outcome were changes of estimated glomerular filtration rate (eGFR) over time. Positive outcome was defined as > 30% increase, stable outcome as ±30%, negative outcome as decrease > 30% of eGFR. RESULTS: Eleven patients (78.8%) were treated with eculizumab, three received standard of care (SoC, 27.2%). Median follow-up time was 68 months (IQR: 45-98 months). Median eculizumab treatment duration was 10 months (IQR 5-46 months). After eculizumab treatment, five patients showed a stable outcome, six patients showed a negative outcome. Among patients receiving SoC, one patient showed a stable outcome, two patients showed a negative outcome. CONCLUSIONS: The benefit of eculizumab in chronic progressive C3 glomerulopathy is limited.
Assuntos
Inativadores do Complemento , Glomerulonefrite Membranoproliferativa , Feminino , Humanos , Complemento C3/análise , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Proteinúria/etiologia , Estudos Retrospectivos , Resultado do Tratamento , Inativadores do Complemento/uso terapêutico , MasculinoRESUMO
The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.
Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Cinesinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Corpos Basais/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Citoesqueleto/metabolismo , Cães , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/metabolismo , Transdução de SinaisRESUMO
The transcription factor NRF2 plays a key role in the protection against environmental stress and maintaining cellular homeostasis. The acetyltransferase p300 is a known component of the NRF2 transcriptional complex and promotes its transcriptional activity. In this study we describe a novel mechanism by which p300 facilitates NRF2 activity. p300 physically interacts with NRF2 and interferes with NRF2-KEAP1 complex formation. In particular, p300 increases NRF2 protein abundance and stability, thereby promoting NRF2 nuclear localization. Notably, the acetyltransferase activity of p300 was indispensable for the stabilizing effects towards NRF2. Furthermore, overexpression of p300 protected HEK293T cells from oxidative stress and increased viability. Together our study uncovers a link between p300 and control of NRF2-KEAP1 signaling via regulation of NRF2 stability and this may act as a novel checkpoint on the adaptation to oxidative stress.
Assuntos
Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fatores de Transcrição de p300-CBP/genética , Adaptação Fisiológica , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Transcrição Gênica , Fatores de Transcrição de p300-CBP/deficiênciaRESUMO
Despite its apparent simplicity, the nematode Caenorhabditis elegans has a high rating as a model in molecular and developmental biology and biomedical research. C. elegans has no excretory system comparable with the mammalian kidney but many of the genes and molecular pathways involved in human kidney diseases are conserved in C. elegans. The plethora of genetic, molecular and imaging tools available in C. elegans has enabled major discoveries in renal research and advanced our understanding of the pathogenesis of genetic kidney diseases. In particular, studies in C. elegans have pioneered the fundamental role of cilia for cystic kidney diseases. In addition, proteins of the glomerular filtration barrier and podocytes are critical for cell recognition, assembly of functional neuronal circuits, mechanosensation and signal transduction in C. elegans. C. elegans has also proved tremendously valuable for aging research and the Von Hippel-Lindau tumor suppressor gene has been shown to modulate lifespan in the nematode. Further, studies of the excretory canal, membrane transport and ion channel function in C. elegans have provided insights into mechanisms of tubulogenesis and cellular homeostasis. This review recounts the way that C. elegans can be used to investigate various aspects of genetic and molecular nephrology. This model system opens up an exciting and new area of study of renal development and diseases.
Assuntos
Caenorhabditis elegans , Modelos Animais de Doenças , Doenças Genéticas Inatas , Doenças Renais Císticas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismoRESUMO
The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle-dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development.
Assuntos
Fator 4 Ativador da Transcrição/genética , Antígenos de Neoplasias/genética , Mutação , Proteínas de Neoplasias/genética , Animais , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Feminino , Ligação Genética , Humanos , Hibridização In Situ , Masculino , Linhagem , Síndrome , Peixe-ZebraRESUMO
BACKGROUND: Polyneuropathy organomegaly endocrinopathy M-protein skin changes (POEMS) syndrome is a rare cause of polyneuropathy. Calciphylaxis, a severe disease leading to necrotic ulcers of the skin, is associated with POEMS syndrome and also with renal disease. This case report describes a patient with POEMS syndrome plus primary focal segmental glomerulosclerosis. CASE PRESENTATION: A 27-year-old Caucasian woman with chronic renal insufficiency due to focal segmental glomerulosclerosis and calciphylaxis presented to our institution with polyneuropathy and encephalopathy. An extensive diagnostic workup revealed POEMS syndrome. Serum concentrations of vascular endothelial growth factor (VEGF) were highly elevated, consistent with POEMS syndrome. CONCLUSION: To our knowledge, this is the first report of a patient with POEMS syndrome and primary focal segmental glomerulosclerosis. The combination of POEMS syndrome, calciphylaxis and primary focal segmental glomerulosclerosis may be coincidental, suggesting the need for additional studies to confirm or exclude this association. VEGF may be an important pathogenetic link, suggesting that treatment with antiangiogenic agents may improve patient outcomes.
Assuntos
Calciofilaxia/fisiopatologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Síndrome POEMS/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Feminino , Humanos , Pele/patologiaRESUMO
Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Desgrenhadas , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt , Proteínas de Xenopus , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The von Hippel-Lindau tumor suppressor gene (VHL) is mutated in up to 90% of clear cell renal cell carcinoma (ccRCC) cases, thus playing a key role in ccRCC pathogenesis. ccRCC can be classified as a metabolic disease in which alterations in fatty acid metabolism facilitate cancer cell proliferation. Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) is an enzyme involved in peroxisomal fatty acid degradation. It is primarily expressed in renal proximal tubule cells, presumably the origin of ccRCC. Although EHHADH is still a relatively unexplored gene, it is known to be differentially expressed in several tumors. In this study, analysis of several databases revealed that EHHADH expression is downregulated in ccRCC samples compared to healthy kidney samples. Moreover, cell culture experiments were performed to investigate the relationship between EHHADH and VHL at the gene and protein level. qPCR and Western blot analyses using the human ccRCC cell line RCC4 revealed that EHHADH is expressed in a VHL-dependent manner. RCC4 cells reconstituted with VHL show significantly higher EHHADH mRNA and protein levels than VHL-deficient RCC4 control cells. These results indicate that the downregulation of EHHADH in ccRCC reported may be due to the loss of VHL function. This study is the first to molecularly characterize EHHADH, a key enzyme in peroxisomal ß-oxidation, in relation to VHL, suggesting a potential pathogenic interaction that is worthy of further investigation.
RESUMO
To maximize the impact of precision medicine approaches, it is critical to identify genetic variants underlying disease and to accurately quantify their functional effects. A gene exemplifying the challenge of variant interpretation is the von Hippel-Lindautumor suppressor (VHL). VHL encodes an E3 ubiquitin ligase that regulates the cellular response to hypoxia. Germline pathogenic variants in VHL predispose patients to tumors including clear cell renal cell carcinoma (ccRCC) and pheochromocytoma, and somatic VHL mutations are frequently observed in sporadic renal cancer. Here we optimize and apply saturation genome editing to assay nearly all possible single-nucleotide variants (SNVs) across VHL's coding sequence. To delineate mechanisms, we quantify mRNA dosage effects and compare functional effects in isogenic cell lines. Function scores for 2,268 VHL SNVs identify a core set of pathogenic alleles driving ccRCC with perfect accuracy, inform differential risk across tumor types and reveal new mechanisms by which variants impact function. These results have immediate utility for classifying VHL variants encountered clinically and illustrate how precise functional measurements can resolve pleiotropic and dosage-dependent genotype-phenotype relationships across complete genes.
Assuntos
Alelos , Carcinoma de Células Renais , Edição de Genes , Neoplasias Renais , Polimorfismo de Nucleotídeo Único , Proteína Supressora de Tumor Von Hippel-Lindau , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Humanos , Edição de Genes/métodos , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Linhagem Celular Tumoral , Predisposição Genética para Doença , MutaçãoRESUMO
Mutations of inversin cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney disease and developmental defects. Inversin regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin is essential for Xenopus pronephros formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin abrogated both proximal pronephros extension and distal tubule differentiation, phenotypes similar to that of Xenopus deficient in Frizzled-8. Exogenous Inversin rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin acts downstream of Frizzled-8 in pronephros morphogenesis. Depletion of Inversin prevents the recruitment of Dishevelled in response to Frizzled-8 and impeded the accumulation of Dishevelled at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/embriologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Desgrenhadas , Fluorescência , Hibridização In Situ , Rim/metabolismo , Camundongos , Microscopia Confocal , Oligonucleotídeos/genética , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , XenopusRESUMO
Mutations of the ankyrin-repeat protein Inversin, a member of a diverse family of more than 12 proteins, cause nephronophthisis (NPH), an autosomal recessive cystic kidney disease associated with extra-renal manifestations such as retinitis pigmentosa, cerebellar aplasia and situs inversus. Most NPH gene products (NPHPs) localize to the cilium, and appear to control the transport of cargo protein to the cilium by forming functional networks. Inversin interacts with NPHP1 and NPHP3, and shares with NPHP4 the ability to antagonize Dishevelled-stimulated canonical Wnt signaling, potentially through recruitment of the Anaphase Promoting Complex (APC/C). However, Dishevelled antagonism may be confined towards the basal body, thereby polarizing motile cilia on the cells of the ventral node and respiratory tract. Inversin is essential for recruiting Dishevelled to the plasma membrane in response to activated Frizzled, a crucial step in planar cell polarity signaling. During vertebrate pronephros development, the Inversin-mediated translocation of Dishevelled appears to orchestrate the migration of cells and differentiation of segments that correspond to the mammalian loop of Henle. Thus, defective tubule migration and elongation may contribute to concentration defects and cause cyst formation in patients with NPH.
Assuntos
Cílios/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Cílios/química , Cílios/metabolismo , Humanos , Rim/citologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Proteínas Wnt/químicaRESUMO
Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer, and inactivation of the VHL tumor suppressor gene is found in almost all cases of hereditary and sporadic ccRCCs. CcRCC is associated with the reprogramming of fatty acid metabolism, and stearoyl-CoA desaturases (SCDs) are the main enzymes controlling fatty acid composition in cells. In this study, we report that mRNA and protein expression of the stearoyl-CoA desaturase SCD5 is downregulated in VHL-deficient cell lines. Similarly, in C. elegans vhl-1 mutants, FAT-7/SCD5 activity is repressed, supporting an evolutionary conservation. SCD5 regulation by VHL depends on HIF, and loss of SCD5 promotes cell proliferation and a metabolic shift towards ceramide production. In summary, we identify a novel regulatory function of VHL in relation to SCD5 and fatty acid metabolism, and propose a new mechanism of how loss of VHL may contribute to ccRCC tumor formation and progression.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Carcinoma de Células Renais/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias Renais/patologia , Proliferação de Células/genética , Homeostase , Lipídeos , Estearoil-CoA Dessaturase/genéticaRESUMO
Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2'-O-methylcytidine. The method's reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Animais , Camundongos , Carcinoma de Células Renais/patologia , Nucleosídeos/uso terapêutico , Reprodutibilidade dos Testes , Transcriptoma , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias Renais/patologia , RNA/uso terapêuticoRESUMO
Planar cell polarity signaling controls a variety of polarized cell behaviors. In multiciliated Xenopus epidermal cells, recruitment of Dishevelled (Dvl) to the basal body and its localization to the center of the ciliary rootlet are required to correctly position the motile cilia. We now report that the anaphase-promoting complex (APC/C) recognizes a D-box motif of Dvl and ubiquitylates Dvl on a highly conserved lysine residue. Inhibition of APC/C function by knockdown of the ANAPC2 subunit disrupts the polarity of motile cilia and alters the directionality of the fluid movement along the epidermis of the Xenopus embryo. Our results suggest that the APC/C activity enables cilia to correctly polarize in Xenopus epidermal cells.
Assuntos
Polaridade Celular/fisiologia , Cílios/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Proteínas de Xenopus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Animais Geneticamente Modificados , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase , Sequência de Bases , Linhagem Celular , Sequência Conservada , Proteínas Desgrenhadas , Células Epidérmicas , Feminino , Marcação de Genes , Humanos , Camundongos , Oligodesoxirribonucleotídeos Antissenso/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Complexos Ubiquitina-Proteína Ligase/deficiência , Complexos Ubiquitina-Proteína Ligase/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/fisiologiaRESUMO
BACKGROUND: ABO-incompatible kidney transplantation (ABOi-KT) is an established way to enlarge the donor pool around the world. Comparability of long-term success and complications to ABO-compatible kidney transplantation (ABOc-KT) are still under debate. METHODS: We evaluated all patients with a living donor kidney transplantation performed between April 1, 2004, and March 31, 2019. RESULTS: A total of 137 ABOi-KT and 346 ABOc-KT were analyzed. We excluded 4 ABOi-KT recipients and 178 ABOc-KT recipients with cyclosporine A-based immunosuppression or without basiliximab induction. Three patients of the ABOi-KT cohort and 6 patients of the ABOc-KT cohort were lost to follow-up and therefore excluded. The patient characteristics were comparable except for the higher age of transplant recipients in the ABOc-KT cohort and longer follow-up of the ABOi-KT cohort. The mean estimated 15-year recipient survival was 89% in the ABOi-KT cohort and 91% in the ABOc-KT cohort (P = .39). Mean estimated graft survival was 71% in the ABOi-KT cohort and 87% in the ABOc-KT cohort (P = .68). The estimated glomerular filtration rate (Modification of Diet in Renal Disease) measured in the last follow-up was 51 mL/min/1.73 m2 in the ABOi-KT cohort and 50 mL/min/1.73 m2 in the ABOc-KT cohort (P = .36). The incidence for antibody-mediated rejection, T cell-mediated rejections, and infectious complications requiring hospitalization was not different between the cohorts. In the ABOi-KT cohort, we found significantly more lymphoceles and consequent surgical revision procedures. CONCLUSIONS: At our center, ABOi-KT has as good long-term results as ABOc-KT in terms of patient survival, graft survival, and complications, with the exception of increased lymphocele formation.
Assuntos
Incompatibilidade de Grupos Sanguíneos/mortalidade , Rejeição de Enxerto/mortalidade , Transplante de Rim/mortalidade , Complicações Pós-Operatórias/mortalidade , Insuficiência Renal Crônica/cirurgia , Adulto , Incompatibilidade de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos/cirurgia , Tipagem e Reações Cruzadas Sanguíneas , Estudos de Coortes , Feminino , Seguimentos , Alemanha , Taxa de Filtração Glomerular , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Terapia de Imunossupressão/métodos , Terapia de Imunossupressão/mortalidade , Transplante de Rim/métodos , Doadores Vivos , Linfocele/imunologia , Linfocele/mortalidade , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/imunologia , Insuficiência Renal Crônica/imunologia , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Inactivation of the tumor suppressor von Hippel-Lindau (VHL) gene is a key event in hereditary and sporadic clear cell renal cell carcinomas (ccRCC). The mechanistic target of rapamycin (mTOR) signaling pathway is a fundamental regulator of cell growth and proliferation, and hyperactivation of mTOR signaling is a common finding in VHL-dependent ccRCC. Deregulation of mTOR signaling correlates with tumor progression and poor outcome in patients with ccRCC. Here, we report that the regulatory-associated protein of mTOR (RAPTOR) is strikingly repressed by VHL. VHL interacts with RAPTOR and increases RAPTOR degradation by ubiquitination, thereby inhibiting mTORC1 signaling. Consistent with hyperactivation of mTORC1 signaling in VHL-deficient ccRCC, we observed that loss of vhl-1 function in C. elegans increased mTORC1 activity, supporting an evolutionary conserved mechanism. Our work reveals important new mechanistic insight into deregulation of mTORC1 signaling in ccRCC and links VHL directly to the control of RAPTOR/mTORC1. This may represent a novel mechanism whereby loss of VHL affects organ integrity and tumor behavior.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Animais , Caenorhabditis elegans , Carcinoma de Células Renais/patologia , Processos de Crescimento Celular/genética , Proliferação de Células/genética , Células HEK293 , Humanos , Neoplasias Renais/patologia , Ubiquitinação/genéticaRESUMO
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Doenças Renais Císticas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/química , Feminino , Técnicas de Silenciamento de Genes , Humanos , Doenças Renais Císticas/complicações , Doenças Renais Císticas/embriologia , Doenças Renais Císticas/genética , Masculino , Microinjeções , Tubo Neural/embriologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
SKN-1/Nrf transcription factors regulate diverse biological processes essentially stress defense, detoxification, and longevity. Studies in model organisms have identified a broad range of regulatory processes and mechanisms that profoundly influence SKN-1/Nrf functions. Defining the mechanisms how SKN-1 is regulated will provide insight how cells defend against diverse stressors contributing to aging and disease. In this study, we demonstrate a crucial role for the acetyltransferase CBP-1, the C. elegans homolog of mammalian CREB-binding protein CBP/p300 in the activation of SKN-1. cbp-1 is essential for tolerance of oxidative stress and normal lifespan. CBP-1 directly interacts with SKN-1 and increases SKN-1 protein abundance. In particular CBP-1 modulates SKN-1 nuclear translocation under basal conditions and in response to stress and promotes SKN-1-dependent transcription of protective genes. Moreover, CBP-1 is required for SKN-1 nuclear recruitment, transcriptional activity, and longevity due to reduced insulin/IGF-1-like signaling, mTOR-, and GSK-3 signaling. Our findings establish the acetyltransferase CBP-1 as a critical activator of SKN-1 that directly modulates SKN-1 protein stability, nuclear localization, and function to ascertain normal stress response and lifespan.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Fatores de Transcrição de p300-CBP/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Longevidade/fisiologia , Estresse Oxidativo/fisiologia , Fatores de Transcrição/genéticaRESUMO
The mechanistic target of rapamycin (mTOR) kinase is central to metabolism and growth, and has a conserved role in aging. mTOR functions in two complexes, mTORC1 and mTORC2. In diverse eukaryotes, inhibition of mTORC1 signaling increases lifespan. mTORC1 transduces anabolic signals to stimulate protein synthesis and inhibits autophagy. In this study, we demonstrate that CGEF-1, the C. elegans homolog of the human guanine nucleotide exchange factor Dbl, is a novel binding partner of RHEB-1 and activator of mTORC1 signaling in C. elegans. cgef-1 mutants display prolonged lifespan and enhanced stress resistance. The transcription factors DAF-16/FoxO and SKN-1/Nrf are required for increased longevity and stress tolerance, and induce protective gene expression in cgef-1 mutants. Genetic evidence indicates that cgef-1 functions in the same pathway with rheb-1, the mTOR kinase let-363, and daf-15/Raptor. When cgef-1 is inactivated, phosphorylation of 4E-BP, a central mTORC1 substrate for protein translation is reduced in C. elegans. Moreover, autophagy is increased upon cgef-1 and mTORC1 inhibition. In addition, we show that in human cells Dbl associates with Rheb and stimulates mTORC1 downstream targets for protein synthesis suggesting that the function of CGEF-1/Dbl in the mTORC1 signaling pathway is evolutionarily conserved. These findings have important implications for mTOR functions and signaling mechanisms in aging and age-related diseases.