RESUMEN
Ras proteins control many aspects of eukaryotic cell homeostasis by switching between active (GTP-bound) and inactive (GDP-bound) conformations, a reaction catalyzed by GTPase exchange factors (GEF) and GTPase activating proteins (GAP) regulators, respectively. Here, we show that the complexity, measured as number of genes, of the canonical Ras switch genetic system (including Ras, RasGEF, RasGAP and RapGAP families) from 24 eukaryotic organisms is correlated with their genome size and is inversely correlated to their evolutionary distances from humans. Moreover, different gene subfamilies within the Ras switch have contributed unevenly to the module's expansion and speciation processes during eukaryote evolution. The Ras system remarkably reduced its genetic expansion after the split of the Euteleostomi clade and presently looks practically crystallized in mammals. Supporting evidence points to gene duplication as the predominant mechanism generating functional diversity in the Ras system, stressing the leading role of gene duplication in the Ras family expansion. Domain fusion and alternative splicing are significant sources of functional diversity in the GAP and GEF families but their contribution is limited in the Ras family. An evolutionary model of the Ras system expansion is proposed suggesting an inherent 'decision making' topology with the GEF input signal integrated by a homologous molecular mechanism and bifurcation in GAP signaling propagation.
Asunto(s)
Evolución Molecular , Proteínas ras/clasificación , Empalme Alternativo , Animales , Variación Genética , Humanos , Mamíferos/genética , Filogenia , Estructura Terciaria de Proteína , Proteínas Activadoras de ras GTPasa/clasificación , Factores de Intercambio de Guanina Nucleótido ras/clasificación , Proteínas ras/química , Proteínas ras/genéticaRESUMEN
The affinities of Ca(2+)-saturated and Ca(2+)-free calmodulin for a fluorescent reporter construct containing the PEP19 IQ domain differ by a factor of approximately 100, with K(d) values of 11.0 +/- 1.2 and 1128.4 +/- 176.5 muM, respectively, while the affinities of a reporter containing the neuromodulin IQ domain are essentially identical, with K(d) values of 2.9 +/- 0.3 and 2.4 +/- 0.3 muM, respectively. When Ca(2+) is bound only to the C-terminal pair of Ca(2+)-binding sites in calmodulin, the K(d) value for the PEP19 reporter complex is decreased approximately 5-fold, while the value for the neuromodulin reporter complex is increased by the same factor. When Ca(2+) is bound only to the N-terminal pair of Ca(2+)-binding sites, the K(d) value for the PEP19 reporter complex is unaffected, but the value for the complex with the neuromodulin reporter is increased approximately 12-fold. These functional differences are largely ascribed to three differences in the CaM-binding sequences of the two reporters. Replacement of a central Gly in the neuromodulin IQ domain with a Lys at this position in PEP19 almost entirely accounts for the distinctive patterns of Ca(2+)-dependent stability changes exhibited by the two complexes. Replacement of a Lys immediately before the "IQ" amino acid pair in the neuromodulin sequence with the Ala in PEP19 accounts for the remaining Ca(2+)-dependent differences. Replacement of an Ala in the N-terminal half of the neuromodulin sequence with the Gln in PEP19 accounts for approximately half of the Ca(2+)-independent difference in the stabilities of the two reporter complexes, with the Ca(2+)-independent effect of the Lys replacement accounting for most of the remainder. Since the central Gly in the neuromodulin sequence is conserved in half of all known IQ domains, these results suggest that the presence or absence of this residue defines two major functional classes.
Asunto(s)
Motivos EF Hand/fisiología , Proteína GAP-43/química , Proteína GAP-43/clasificación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/clasificación , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/clasificación , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Calmodulina/fisiología , Motivos EF Hand/genética , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Genes Reporteros/fisiología , Glicina/genética , Humanos , Ligandos , Lisina/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/genética , Estabilidad Proteica , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Capillary malformation-arteriovenous malformation (CM-AVM) is a newly discovered hereditary disorder. Its defining features are atypical cutaneous multifocal capillary malformations often in association with high-flow lesions: cutaneous, subcutaneous, intramuscular, intraosseous and cerebral arteriovenous malformations and arteriovenous fistulas. Some patients have Parkes Weber syndrome - a large congenital cutaneous vascular stain in an extremity, with bony and soft tissue hypertrophy and microscopic arteriovenous shunting. In the past, arteriovenous malformations and arteriovenous fistulas had been considered non-hereditary. A classical genetic approach was used to identify the locus. Candidate gene screening pinpointed mutations in RASA1 (p120-RASGAP) - a RasGTPase. RASA1 reverts active GTP-bound Ras into inactive GDP-bound form. Murine Rasa1 knockout and tetraploid-aggregated embryos with RNA interference exhibited abnormal vascular development. Lack of RASA1 activity caused inhibition of cell motility, possibly through p190-RhoGAP. Thus, RASA1 defects probably cause abnormal angiogenic remodeling of the primary capillary plexus that cannot be compensated for by other RasGAPs: RASA2, RASAL and NF1. Signaling pathways involving RASA1 might offer novel targets for treatment of high-flow vascular anomalies.