RESUMEN
Oculoectodermal syndrome (OES) and encephalocraniocutaneous lipomatosis (ECCL) are rare disorders that share many common features, such as epibulbar dermoids, aplasia cutis congenita, pigmentary changes following Blaschko lines, bony tumor-like lesions, and others. About 20 cases with OES and more than 50 patients with ECCL have been reported. Both diseases were proposed to represent mosaic disorders, but only very recently whole-genome sequencing has led to the identification of somatic KRAS mutations, p.Leu19Phe and p.Gly13Asp, in affected tissue from two individuals with OES. Here we report the results of molecular genetic studies in three patients with OES and one with ECCL. In all four cases, Sanger sequencing of the KRAS gene in DNA from lesional tissue detected mutations affecting codon 146 (p.Ala146Val, p.Ala146Thr) at variable levels of mosaicism. Our findings thus corroborate the evidence of OES being a mosaic RASopathy and confirm the common etiology of OES and ECCL. KRAS codon 146 mutations, as well as the previously reported OES-associated alterations, are known oncogenic KRAS mutations with distinct functional consequences. Considering the phenotype and genotype spectrum of mosaic RASopathies, these findings suggest that the wide phenotypic variability does not only depend on the tissue distribution but also on the specific genotype.
Asunto(s)
Quiste Dermoide/genética , Displasia Ectodérmica/genética , Oftalmopatías/genética , Predisposición Genética a la Enfermedad , Lipomatosis/genética , Síndromes Neurocutáneos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Niño , Preescolar , Codón , Quiste Dermoide/patología , Displasia Ectodérmica/patología , Oftalmopatías/patología , Humanos , Lactante , Lipomatosis/patología , Síndromes Neurocutáneos/patologíaRESUMEN
BACKGROUND: Platelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested in vivo for a variety of disorders. Two small-molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively. OBJECTIVES: To analyze the specificity of NSC23766 and EHT1864. METHODS: Platelet function was assessed in mouse wild-type and Rac1-deficient platelets by the use of flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed with differential interference contrast microscopy, and activation of effector molecules was analyzed with biochemical approaches. RESULTS: NSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100 µm in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoprotein Ib-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors p21-activated kinase (PAK)1 and PAK2. CONCLUSIONS: Our results reveal critical off-target effects of NSC23766 and EHT1864 at 100 µm in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.
Asunto(s)
Aminoquinolinas/farmacología , Plaquetas/efectos de los fármacos , Neuropéptidos/antagonistas & inhibidores , Pirimidinas/farmacología , Pironas/farmacología , Quinolinas/farmacología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Plaquetas/metabolismo , Ratones , Ratones Noqueados , Neuropéptidos/genética , Fosforilación , Transducción de Señal , Proteína de Unión al GTP rac1/genéticaRESUMEN
The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.
Asunto(s)
Mutación de Línea Germinal , Herpesvirus Humano 4/fisiología , Trastornos Linfoproliferativos/virología , Proteínas Tirosina Quinasas/genética , Sitios de Unión , Niño , Preescolar , Femenino , Humanos , Masculino , Mutación Missense , Linaje , Fosforilación , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Noonan and Cardio-facio-cutaneous (CFC) syndromes are characterized by typical dysmorphic features, cardiac defects, short stature, variable ectodermal anomalies, and intellectual disability. Both belong to the Ras/mitogen-activated protein kinase pathway group of disorders and clinical features overlap other related conditions, notably LEOPARD and Costello syndromes. KRAS mutations account for about 2% of reported Noonan and <5% of reported CFC cases. The mutation spectrum includes recurrent missense changes clustering in particular domains of the KRAS protein and conferring gain-of-function. We report three patients from two unrelated families with novel missense KRAS mutations, p.K147E and p.Y71H. Both mutations affect a residue which is highly conserved in KRAS and other RAS isoforms. One of the families includes a mother and son pair who represent the first report of a vertically transmitted KRAS mutation. In addition, the mother and son pair had peripheral neuropathy, complicated by Charcot arthropathy in the mother. An unusual phenotypic effect of the specific KRAS mutation or a coincidence of two independent disorders may be considered. KRAS mutation-associated phenotypes appear to be subject to considerable clinical heterogeneity. All three cases highlight the challenges of clinical assessment in KRAS mutation-positive patients, and the utility of molecular testing as an adjunct to diagnosis.
Asunto(s)
Mutación de Línea Germinal , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Artropatía Neurógena/complicaciones , Artropatía Neurógena/genética , Preescolar , Diagnóstico Diferencial , Displasia Ectodérmica/complicaciones , Displasia Ectodérmica/genética , Facies , Insuficiencia de Crecimiento/complicaciones , Insuficiencia de Crecimiento/genética , Femenino , Cardiopatías Congénitas/complicaciones , Cardiopatías Congénitas/genética , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , Síndrome de Noonan/genética , Linaje , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/genética , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G1 through a cellular senescence program characterized by the upregulation of nuclear p21(WAF), downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1γ (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated ß-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Senescencia Celular/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/fisiología , Paclitaxel/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Doxorrubicina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Microtúbulos/efectos de los fármacosRESUMEN
OBJECTIVE: To measure HIV prevalence and characterize associated risk behaviors among injection drug users (IDU) upon detention in Tehran, Iran. METHODS: A cross-sectional survey included 459 male IDU arrested by police during a police sweep in Tehran in 2006. A questionnaire was completed, and blood was collected for HIV testing. RESULTS: Overall HIV prevalence was 24.4% (95% confidence interval 20.5-28.6). Factors independently associated with HIV infection included history of using an opioid in jail (adjusted odds ratio 2.11, 95% confidence interval 1.26-3.53) and older age (adjusted odds ratio 2.79 for 25-34, 3.01 for 35-44, 4.62 for > or = 45 yr). CONCLUSIONS: This study supports that incarceration is contributing to the increased spread of HIV. Harm reduction programs should be urgently expanded, particularly among incarcerated IDU.
Asunto(s)
Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , Abuso de Sustancias por Vía Intravenosa/complicaciones , Adolescente , Adulto , Estudios Transversales , Humanos , Irán/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Factores de Riesgo , Adulto JovenRESUMEN
The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (P(i)) by guanine nucleotide binding proteins (GNBPs) is a fundamental process in living cells and represents an important timer in intracellular signalling and transport processes. While the rate of GNBP-mediated GTP hydrolysis is intrinsically slow, direct interaction with GTPase activating proteins (GAPs) accelerates the reaction by up to five orders of magnitude in vitro. Eighteen years after the discovery of the first GAP, biochemical and structural research has been accumulating evidence that GAPs employ a much wider spectrum of chemical mechanisms than had originally been assumed, in order to regulate the chemical players on the catalytic protein-protein interaction stage.
Asunto(s)
Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/fisiología , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. Here we present the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its 'switch' regions for interacting with two subdomains of GBD/FH3. We show that the FH3 domain of mDia1 forms a stable dimer and we also identify the DAD-binding site. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of our results, we propose a structural model for the regulation of mDia1 by Rho and DAD.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Portadoras/genética , Dimerización , Forminas , Células HeLa , Humanos , Mamíferos , Modelos Moleculares , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteínas de Unión al GTP rho/genéticaRESUMEN
The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Catálisis , Escherichia coli , Genes ras , Unión Proteica , Especificidad por SustratoRESUMEN
Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.
Asunto(s)
Genes ras/genética , Melanoma/genética , Mutación Puntual , Codón , ADN de Neoplasias/genética , Exones , Femenino , Guanilil Imidodifosfato/metabolismo , Humanos , Masculino , Melanoma/patología , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Estudios Retrospectivos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Regulación hacia Abajo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas de Unión al GTP rac/metabolismo , Compuestos de Aluminio/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Fluoruros/metabolismo , Proteínas Activadoras de GTPasa/química , Guanosina Difosfato/metabolismo , Histidina Quinasa , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/patogenicidad , Alineación de Secuencia , Relación Estructura-Actividad , Proteínas de Unión al GTP rac/químicaRESUMEN
Rho family GTPases control numerous cellular processes including cytoskeletal reorganization and transcriptional activation. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) which stimulate the exchange of bound GDP for GTP. We recently isolated a putative GEF, termed p190RhoGEF that binds to RhoA and, when overexpressed in neuronal cells, induces cell rounding and inhibits neurite outgrowth. Here we show that the isolated tandem Dbl homology/pleckstrin homology domain of p190RhoGEF activates RhoA in vitro, but not Rac1 or Cdc42, as determined by GDP release and protein binding assays. In contrast, full-length p190RhoGEF fails to activate RhoA in vitro. When overexpressed in intact cells, however, p190RhoGEF does activate RhoA with subsequent F-actin reorganization and serum response factor-mediated transcription. Immunofluorescence studies show that endogenous p190RhoGEF localizes to distinct RhoA-containing regions at the plasma membrane, to the cytosol and along microtubules. In vitro and in vivo binding experiments show that p190RhoGEF directly interacts with microtubules via its C-terminal region adjacent to the catalytic Dbl homology/pleckstrin homology domain. Our results indicate that p190RhoGEF is a specific activator of RhoA that requires as yet unknown binding partners to unmask its GDP/GTP exchange activity in vivo, and they suggest that p190RhoGEF may provide a link between microtubule dynamics and RhoA signaling.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Animales , Células COS , Compartimento Celular , Línea Celular , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas Nucleares/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Respuesta Sérica , Fibras de Estrés/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Tubulina (Proteína)/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The small guanosine triphosphate (GTP) binding protein Ras is involved in many cellular signal transduction processes leading to cell growth, differentiation and apoptosis. Mutations in ras genes are found in a large number of human tumours. GTP hydrolysis, the process that normally leads to the transition of the Ras protein from the active (GTP-bound) form to the inactive (GDP-bound) form is impaired due to these oncogenic mutations. In contrast, the GTP analogue 3,4-diaminobenzophenone(DABP)-phosphoramidate-GTP, a substrate for GTP-binding proteins, enables switching to the inactive GDP form in both wild-type and oncogenic Ras. Here we show by HPLC, mass spectrometry and NMR spectroscopy that the mechanism of this DABP-GTPase reaction is different from the physiological GTPase reaction. The gamma-phosphate group is not attacked by a nucleophilic water molecule, but rather by the aromatic amino group of the analogue, which leads to the generation of a stable cyclic diamidate product. These findings have potential implications for the development of anti-Ras drugs.
Asunto(s)
Benzofenonas/química , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Proteínas ras/química , Benzofenonas/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Resonancia Magnética Nuclear Biomolecular , Proteínas ras/metabolismoRESUMEN
Mutations of Ras with three extra amino acids inserted into the phosphate-binding (P) loop have been investigated both in vitro and in vivo. Such mutants have originally been detected as oncogenes both in the ras and the TC21 genes. Biochemical experiments reveal the molecular basis of their oncogenic potential: the mutants show a strongly attenuated binding affinity for nucleotides, most notably for GDP, leading to a preference for GTP binding. Furthermore, both the intrinsic as well as the GAP-stimulated GTP hydrolysis are drastically diminished. The binding interaction with GAP is reduced, whereas binding to the Ras-binding domain of the downstream effector c-Raf1 is not altered appreciably. Microinjection into PC12 cells shows the mutants to be as potent to induce neurite outgrowth as conventional oncogenic Ras mutants. Unexpectedly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells is much higher than that of the normal oncogenic mutant G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutants.
Asunto(s)
Guanosina Trifosfato/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Animales , Sitios de Unión , Clonación Molecular , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Genes ras , Mutagénesis Insercional , Nucleótidos , Proteína Oncogénica p21(ras)/genética , Células PC12 , Fosfatos/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Conejos , RatasRESUMEN
Guanine nucleotide exchange factors of the Dbl family regulate the actin cytoskeleton through activation of Rho-like GTPases. At present the Dbl family consists of more than thirty members; many have not been phenotypically or biochemically characterized. Guanine nucleotide exchange factors universally feature a Dbl homology domain followed by a pleckstrin homology domain. Employing data base screening we identified a recently cloned cDNA, KIAA0424, showing substantial sequence homology with Rac activators such as Tiam1, Sos, Vav, and PIX within the catalytic domain. This cDNA appears to be the human homologue of the Ascidian protein Posterior End Mark-2 (PEM-2). We refer to this exchanger as hPEM-2. hPEM-2 encodes a protein of 70 kDa and features an N-terminal src homology 3 domain, followed by tandem Dbl homology and pleckstrin homology domains. The gene is highly expressed in brain and is localized on the human X-chromosome. Employing biochemical activity assays for Rho-like GTPases we found that hPEM-2 specifically activates Cdc42 and not Rac or RhoA. Ectopic expression of hPEM-2 in NIH3T3 fibroblasts revealed a Cdc42 phenotype featuring filopodia formation, followed by cortical actin polymerization and cell rounding. hPEM-2 represents an exchange factor, which may have a role in the regulation of a number of cellular processes through Cdc42.
Asunto(s)
Proteínas del Huevo/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Proteínas del Huevo/química , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Fenotipo , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.
Asunto(s)
Benzofenonas/análisis , Genes ras , Guanosina Trifosfato/análogos & derivados , Mutación , Proteínas ras/genética , Proteínas ras/metabolismo , Benzofenonas/química , Benzofenonas/metabolismo , Cristalografía por Rayos X , Escherichia coli , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/análisis , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Stimulation of the intrinsic GTPase activity of GTP-binding proteins by GTPase-activating proteins (GAPs) is a basic principle of GTP-binding-protein downregulation. Recently, the molecular mechanism behind this reaction has been elucidated by studies on Ras and Rho, and their respective GAPs. The basic features involve stabilizing the existing catalytic machinery and supplementing it by an external arginine residue. This represents a novel mechanism for enzyme active-site formation.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Activación Enzimática , Proteínas Activadoras de GTPasa , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Proteínas/química , Proteínas/genética , Homología de Secuencia de Aminoácido , Proteínas Activadoras de ras GTPasaRESUMEN
Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most frequent genetic disorders in man. It acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which despite very limited sequence homology is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed which is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions.
Asunto(s)
Estructura Terciaria de Proteína , Proteínas/química , Empalme Alternativo , Secuencia de Aminoácidos , Catálisis , Cristalografía por Rayos X , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/química , Neurofibromina 1 , Unión Proteica , Estructura Secundaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismo , Proteínas Activadoras de ras GTPasa , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, café-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras.GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.
Asunto(s)
Genoma Humano , Mutación , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Femenino , Proteínas Activadoras de GTPasa , Regulación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Neurofibromina 1 , Alineación de Secuencia , Proteínas Activadoras de ras GTPasaRESUMEN
Monoglucosylation of low molecular mass GTPases is an important post-translational modification by which microbes interfere with eukaryotic cell signaling. Ha-Ras is monoglucosylated at effector domain amino acid threonine 35 by Clostridium sordellii lethal toxin, resulting in a blockade of the downstream mitogen-activated protein kinase cascade. To understand the molecular consequences of this modification, effects of glucosylation on each step of the GTPase cycle of Ras were analyzed. Whereas nucleotide binding was not significantly altered, intrinsic GTPase activity was markedly decreased, and GTPase stimulation by the GTPase-activating protein p120(GAP) and neurofibromin NF-1 was completely blocked, caused by failure to bind to glucosylated Ras. Guanine nucleotide exchange factor (Cdc25)-catalyzed GTP loading was decreased, but not completely inhibited. A dominant-negative property of modified Ras to sequester exchange factor was not detectable. However, the crucial step in downstream signaling, Ras-effector coupling, was completely blocked. The Kd for the interaction between Ras.GTP and the Ras-binding domain of Raf was 15 nM, whereas glucosylation increased the Kd to >1 mM. Because the affinity of Ras.GDP for Raf (Kd = 22 microM) is too low to allow functional interaction, a glucose moiety at threonine 35 of Ras seems to block completely the interaction with Raf. The net effect of lethal toxin-catalyzed glucosylation of Ras is the complete blockade of Ras downstream signaling.