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1.
PLoS One ; 18(8): e0289778, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37540694

RESUMO

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.


Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Camundongos , Animais , Canais de Cátion TRPP/química , Rim Policístico Autossômico Dominante/genética , Modelos Animais de Doenças
2.
Sci Rep ; 8(1): 2743, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29426897

RESUMO

Recent studies have reported intrinsic metabolic reprogramming in Pkd1 knock-out cells, implicating dysregulated cellular metabolism in the pathogenesis of polycystic kidney disease. However, the exact nature of the metabolic changes and their underlying cause remains controversial. We show herein that Pkd1 k o /ko renal epithelial cells have impaired fatty acid utilization, abnormal mitochondrial morphology and function, and that mitochondria in kidneys of ADPKD patients have morphological alterations. We further show that a C-terminal cleavage product of polycystin-1 (CTT) translocates to the mitochondria matrix and that expression of CTT in Pkd1 ko/ko cells rescues some of the mitochondrial phenotypes. Using Drosophila to model in vivo effects, we find that transgenic expression of mouse CTT results in decreased viability and exercise endurance but increased CO2 production, consistent with altered mitochondrial function. Our results suggest that PC1 may play a direct role in regulating mitochondrial function and cellular metabolism and provide a framework to understand how impaired mitochondrial function could be linked to the regulation of tubular diameter in both physiological and pathological conditions.


Assuntos
Rim , Mitocôndrias , Proteínas Mitocondriais/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteólise , Canais de Cátion TRPP/metabolismo , Idoso , Animais , Animais Geneticamente Modificados , Cães , Drosophila melanogaster , Embrião de Mamíferos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Ácidos Graxos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Canais de Cátion TRPP/genética
3.
Sci Rep ; 7(1): 7733, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798345

RESUMO

Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-ß signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-ß signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/deficiência , Animais , Biomarcadores , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Mutação , Rim Policístico Autossômico Recessivo/patologia , Transporte Proteico , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
4.
EBioMedicine ; 5: 183-92, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27077126

RESUMO

BACKGROUND: The major gene mutated in autosomal dominant polycystic kidney disease was first identified over 20 years ago, yet its function remains poorly understood. We have used a systems-based approach to examine the effects of acquired loss of Pkd1 in adult mouse kidney as it transitions from normal to cystic state. METHODS: We performed transcriptional profiling of a large set of male and female kidneys, along with metabolomics and lipidomics analyses of a subset of male kidneys. We also assessed the effects of a modest diet change on cyst progression in young cystic mice. Fatty acid oxidation and glycolytic rates were measured in five control and mutant pairs of epithelial cells. RESULTS: We find that females have a significantly less severe kidney phenotype and correlate this protection with differences in lipid metabolism. We show that sex is a major determinant of the transcriptional profile of mouse kidneys and that some of this difference is due to genes involved in lipid metabolism. Pkd1 mutant mice have transcriptional profiles consistent with changes in lipid metabolism and distinct metabolite and complex lipid profiles in kidneys. We also show that cells lacking Pkd1 have an intrinsic fatty acid oxidation defect and that manipulation of lipid content of mouse chow modifies cystic disease. INTERPRETATION: Our results suggest PKD could be a disease of altered cellular metabolism.


Assuntos
Ácidos Graxos/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Transgênicos , Oxirredução , Rim Policístico Autossômico Dominante/fisiopatologia , Canais de Cátion TRPP/metabolismo
5.
J Cell Biol ; 185(3): 459-73, 2009 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-19414608

RESUMO

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Poro Nuclear/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Deleção de Genes , Genoma Fúngico , Metionina/deficiência , Membrana Nuclear/genética , Membrana Nuclear/fisiologia , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
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