RESUMEN
Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
Asunto(s)
Enfermedades Renales Quísticas/genética , Proteínas de la Membrana/genética , Transducción de Señal , Animales , Ataxina-10 , Centrosoma/metabolismo , Cilios/metabolismo , Trastornos de la Motilidad Ciliar/genética , Encefalocele/genética , Proteínas Hedgehog/metabolismo , Humanos , Enfermedades Renales Quísticas/metabolismo , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Enfermedades Renales Poliquísticas/genética , Retinitis Pigmentosa , Pez CebraRESUMEN
The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic ß sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Caenorhabditis elegans , Cilios/metabolismo , GTP Fosfohidrolasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cinesinas , Proteínas de Unión al GTP Monoméricas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular , Cilios/enzimología , GTP Fosfohidrolasas/genética , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Cellular quiescence, defined as reversible growth/proliferation arrest, is thought to represent a homogenous state induced by diverse anti-mitogenic signals. We used transcriptional profiling to characterize human diploid fibroblasts that exited the cell cycle after exposure to three independent signals--mitogen withdrawal, contact inhibition, and loss of adhesion. We show here that each signal caused regulation of a unique set of genes known to be important for cessation of growth and division. Therefore, contrary to expectation, cells enter different quiescent states that are determined by the initiating signal. However, underlying this diversity we discovered a set of genes whose specific expression in non-dividing cells was signal-independent, and therefore representative of quiescence per se, rather than the signal that induced it. This fibroblast "quiescence program" contained genes that enforced the non-dividing state, and ensured the reversibility of the cell cycle arrest. We further demonstrate that one mechanism by which the reversibility of quiescence is insured is the suppression of terminal differentiation. Expression of the quiescence program was not simply a downstream consequence of exit from the cell cycle, because key parts, including those involved in suppressing differentiation, were not recapitulated during the cell cycle arrest caused by direct inhibition of cyclin-dependent kinases. These studies form a basis for understanding the normal biology of cellular quiescence.
Asunto(s)
Fase de Descanso del Ciclo Celular/fisiología , Adhesión Celular/genética , Diferenciación Celular , Línea Celular , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Humanos , Transducción de Señal , Factores de TiempoRESUMEN
Quiescent and tumor cells share the ability to evade irreversible cell fates. Recent studies have shown that the transcriptional regulator Hairy and Enhancer of Split 1 (HES1) protects quiescent fibroblasts from differentiation or senescence. HES1 is highly expressed in rhabdomyosarcomas, and the inhibition of HES1 restores differentiation in these cells. Pathways that lead to elevated HES1 levels, such as the Notch and Hedgehog pathways, are frequently upregulated in tumors. Compounds that inhibit these pathways induce differentiation and apoptosis in cancer cells and several are in clinical trials. HES1 might repress gene expression in part by recruiting histone deacetylases (HDACs). HDACs inhibit differentiation, whereas histone deacetylase inhibitors (HDACis) induce differentiation or apoptosis in tumors and are also showing promise as therapeutics. Small molecules that directly target HES1 itself were recently identified. Here, we discuss the importance of HES1 function in quiescent and tumor cells. Elucidating the pathways that control quiescence could provide valuable information not only for treating cancer but also other diseases.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Neoplasias/fisiopatología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Factor de Transcripción HES-1RESUMEN
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
Asunto(s)
Autoantígenos/genética , Exones/genética , Estudios de Asociación Genética , Enfermedades Renales/genética , Mutación/genética , Proteínas de Neoplasias/genética , Enfermedades de la Retina/genética , Animales , Western Blotting , Estudios de Casos y Controles , Centrosoma/metabolismo , AMP Cíclico/metabolismo , Familia , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Humanos , Enfermedades Renales/patología , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestructura , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ratas , Enfermedades de la Retina/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Técnicas del Sistema de Dos Híbridos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
The cellular state of quiescence is characterized by an exit from the cell cycle that is reversible, that is, upon appropriate stimulation, quiescent cells can re-enter the cell cycle, proliferate and produce progeny. In this way, quiescent cells can be distinguished from cells in an irreversibly arrested state such as senescence or terminal differentiation. The molecular basis for reversible versus irreversible cell cycle arrest is unclear. In a recent study, we demonstrated that the transcriptional regulator Hes1 has a role in maintaining fibroblasts in a reversible quiescent state: overexpression of Hes1 protects fibroblasts against senescence or differentiation, and inhibition of endogenous Hes1 makes quiescent fibroblasts more susceptible to these states. Here we describe the molecular mechanisms by which Hes1 regulates gene expression by modifying histone tails and thus affecting chromatin conformation. We put forward models for how Hes1 is regulated and how it protects quiescent cells from differentiation and senescence.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Fibroblastos/citología , Proteínas de Homeodominio/metabolismo , Envejecimiento , Diferenciación Celular , Línea Celular , Senescencia Celular , Fibroblastos/metabolismo , Humanos , Receptores Notch/metabolismo , Fase de Descanso del Ciclo Celular , Transducción de Señal , Sirtuina 1 , Sirtuinas/metabolismo , Factor de Transcripción HES-1RESUMEN
The mechanisms by which quiescent cells, including adult stem cells, preserve their ability to resume proliferation after weeks or even years of cell cycle arrest are not known. We report that reversibility is not a passive property of nondividing cells, because enforced cell cycle arrest for a period as brief as 4 days initiates spontaneous, premature, and irreversible senescence. Increased expression of the gene encoding the basic helix-loop-helix protein HES1 was required for quiescence to be reversible, because HES1 prevented both premature senescence and inappropriate differentiation in quiescent fibroblasts. In some human tumors, the HES1 pathway was activated, which allowed these cells to evade differentiation and irreversible cell cycle arrest. We conclude that HES1 safeguards against irreversible cell cycle exit both during normal cellular quiescence and pathologically in the setting of tumorigenesis.