RESUMEN
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
Asunto(s)
Citoesqueleto/genética , Proteínas Activadoras de GTPasa/genética , Integrinas/genética , Mecanotransducción Celular/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína de Unión al GTP rac1/genética , Animales , Células COS , Adhesión Celular , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Biología Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Perros , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Pan troglodytes , Dominios Proteicos , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/clasificación , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1. A full-length mouse expressed sequence tag was expressed in human MPS IIIC fibroblasts, and its protein product localized to the lysosome and corrected the enzymatic defect. The mouse sequence was used to identify the full-length human homolog (HGSNAT), which encodes a protein with no homology to other proteins of known function but is highly conserved among plants and bacteria. Mutational analyses of two MPS IIIC cell lines identified a splice-junction mutation that accounted for three mutant alleles, and a single base-pair insertion accounted for the fourth.
Asunto(s)
Acetiltransferasas/genética , Mucopolisacaridosis III/genética , Regiones no Traducidas 3' , Acetiltransferasas/química , Acetiltransferasas/deficiencia , Secuencia de Aminoácidos , Animales , Exones , Etiquetas de Secuencia Expresada , Fibroblastos , Mutación del Sistema de Lectura , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Intrones , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mucopolisacaridosis III/enzimología , Proteínas , Proteómica , Sitios de Empalme de ARN , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , TransfecciónRESUMEN
Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas , Fraccionamiento Celular/métodos , Endosomas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Microscopía Inmunoelectrónica , Modelos Biológicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Polietilenglicoles , Presenilina-1 , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date. We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.