ABSTRACT
This paper reports the EhGEF1-EhRacG and EhGEF1-EhRho1 molecular complexes from Entamoeba histolytica. The not conserved amino acids Gln201,Tyr299, Gln302, Lys312, Asn313, Phe314 and Ile324 were localized, by means of an in silico computational analysis, at the interface of the exposed face from the DH domain of EhGEF1, which are important to establish the contact with its target GTPases EhRacG and EhRho1. Functional studies of nucleotide exchange of Phe314Ala mutant showed a decrement of 80% on EhRacG GTPase; in contrast the Ile324Ala mutant exhibited a reduction of 77%, specifically on EhRho1; meanwhile the Gln302Ala mutant showed a reduction of approximately 50% on the exchange activity for both GTPases. Moreover, the functional studies of the protein EhGEF1 mutants in the conserved residues Thr194Ala, Asn366Ala and Glu367Ala indicated that contrary to what has been reported for other systems, the mutation of these residues did not alter considerably its catalytic activity.
Subject(s)
Chloride Channels/chemistry , Chloride Channels/genetics , Entamoeba histolytica/physiology , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Chloride Channels/metabolism , DNA Mutational Analysis , Entamoeba histolytica/genetics , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Mutation, Missense , Protein Binding , Protein Interaction Mapping , Protein Structure, Quaternary , Protozoan Proteins/metabolismABSTRACT
Astrocytomas develop intense vascular proliferation, essential for tumour growth and invasiveness. Angiotensin II (ANGII) was initially described as a vasoconstrictor; recent studies have shown its participation in cellular proliferation, vascularisation, and apoptosis. We conducted a prospective study to evaluate the expression of ANGII receptors - AT1 and AT2 - and their relationship with prognosis. We studied 133 tumours from patients with diagnosis of astrocytoma who underwent surgery from 1997 to 2002. AT1 and AT2 were expressed in 52 and 44% of the tumours, respectively, when determined by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Ten per cent of low-grade astrocytomas were positive for AT1, whereas grade III and IV astrocytomas were positive in 67% (P<0.001). AT2 receptors were positive in 17% of low-grade astrocytomas and in 53% of high-grade astrocytomas (P=0.01). AT1-positive tumours showed higher cellular proliferation and vascular density. Patients with AT1-positive tumours had a lower survival rate than those with AT1-negative (P<0.001). No association to survival was found for AT2 in the multivariate analysis. Expression of AT1 and AT2 is associated with high grade of malignancy, increased cellular proliferation, and angiogenesis, and is thus related to poor prognosis. These findings suggest that ANGII receptors might be potential therapeutic targets for high-grade astrocytomas.
