RESUMEN
Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of Ocrl in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global Ocrl-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.
Asunto(s)
Túbulos Renales Proximales , Mutación , Síndrome Oculocerebrorrenal/genética , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Animales , Humanos , Ratones , Ratones NoqueadosRESUMEN
The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.
Asunto(s)
Endocitosis , Endosomas/enzimología , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones Noqueados , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.
Asunto(s)
Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Fibroblastos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Células Cultivadas , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Endocitosis/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente/métodos , Mutación , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/patología , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Interferencia de ARN , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.
Asunto(s)
Dermis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Tiadiazoles/farmacología , Triazoles/farmacología , Células Cultivadas , Dermis/citología , Dermis/enzimología , Ensayo de Cambio de Movilidad Electroforética , Inhibidores Enzimáticos/química , Fibroblastos/citología , Fibroblastos/enzimología , Polarización de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Inositol Polifosfato 5-Fosfatasas , Estructura Molecular , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Colorantes de Rosanilina , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Tiadiazoles/química , Triazoles/químicaRESUMEN
Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or ß1 integrin, or the α3ß1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in ß1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.