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1.
J Am Soc Nephrol ; 32(3): 563-579, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33514561

RESUMO

BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Podócitos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Animais , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Podócitos/ultraestrutura , Mapeamento de Interação de Proteínas , Proteoma , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Transcriptoma
2.
Proc Natl Acad Sci U S A ; 114(23): E4621-E4630, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28536193

RESUMO

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.


Assuntos
Actomiosina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Animais , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Feminino , Adesões Focais/patologia , Técnicas de Inativação de Genes , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Podócitos/patologia , Gravidez , Proteômica , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais
3.
Am J Pathol ; 186(2): 324-36, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26683665

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Complexos Multiproteicos/efeitos dos fármacos , Glândulas Seminais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Animais , Masculino , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Transgênicos , Fosforilação , Glândulas Seminais/metabolismo , Fatores de Transcrição/metabolismo
4.
Cells ; 12(9)2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37174721

RESUMO

Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.


Assuntos
Miopatias Congênitas Estruturais , Agregados Proteicos , Humanos , Filaminas/genética , Filaminas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
5.
J Clin Invest ; 133(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37014703

RESUMO

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.


Assuntos
Doença de Fabry , Podócitos , Humanos , Podócitos/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Doença de Fabry/genética , Doença de Fabry/tratamento farmacológico , Doença de Fabry/patologia , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , alfa-Galactosidase/uso terapêutico , Rim/metabolismo , Triexosilceramidas/metabolismo , Triexosilceramidas/farmacologia , Triexosilceramidas/uso terapêutico
6.
Neurol Genet ; 7(3): e590, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34235269

RESUMO

OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

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