The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis

The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.     

An Association Analysis of Reelin Gene (<Emphasis Type="Italic">RELN</Emphasis>) Polymorphisms with Childhood Epilepsy in Eastern Indian Population from West Bengal

Epilepsy is a common neurological condition characterized by unprovoked seizure attacks. Early brain developmental abnormalities involving neuronal migration and lamination are implicated in childhood epilepsy. Reelin, a neuronal-signaling molecule plays a crucial role in these migratory processes. Therefore, reelin gene (RELN), which is located on human chromosome 7q22 is considered as a potential candidate gene for childhood epilepsy. In this study, we recruited 63 patients with childhood-onset epilepsy and 103 healthy controls from West Bengal in India. Genomic DNA isolated from leukocytes of cases and control individuals were used for genotyping analysis of 16 markers of RELN. Case–control analysis revealed significant over-representation of G/C and (G/C+C/C) genotypes, and C allele of exon 22 G/C marker (rs362691) in cases as compared to controls. Pair-wise linkage disequilibrium analysis demonstrated two separate LD blocks with moderately high D′ values in epileptic cases. Based on these data, we have carried out haplotype case–control analysis. Even though we found over-representation of A-C haplotype of intron 12 A/C/exon 22 G/C markers and haplotype combination involving G-allele of exon 22 marker in cases and controls, respectively, the overall test was not significant. LD in this region involving this marker was also more robust in epileptic cases. Taken together, the results provide possible evidences for association of exon 22 G/C marker or any marker in the vicinity, which is in LD with this marker with epilepsy in the West Bengal population. Further investigations involving higher sample sizes are warranted to validate the present finding.     

Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.     

Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms with Coronary Artery Disease: An Updated Meta-Analysis and Systematic Review

Several association studies of endothelial nitric oxide synthase (NOS3) gene polymorphisms with respect to coronary artery disease (CAD) have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far (89 articles, 132 separate studies, and a sample size of 69,235), examining association of three polymorphic forms of the NOS3 gene (i.e. Glu298Asp, T786-C and 27bp VNTR b/a) with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association (OR range = 1.28–1.52, and P<0.00001 for all comparisons), followed by T786-C (OR range = 1.34–1.42, and P<0.00001 for all comparisons) and 4b/a, (OR range = 1.19–1.41, and P≤0.002 for all comparisons) in our pooled analysis. Subgroup analysis revealed that Glu298Asp (OR range = 1.54–1.87, and P<0.004 for all comparisons) and 4b/a (OR range = 1.71–3.02, and P<0.00001 for all comparisons) have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry (OR range = 1.61–1.90, and P≤0.01 for all comparisons).