Association of miR-149 T>C and miR-196a2 C>T Polymorphisms with Colorectal Cancer Susceptibility: A Case-Control Study.

The principal aim of the current study was to investigate the relationship between miR-149 T>C (rs2292832) and miR-196a2 C>T (rs11614913) small non-coding RNA polymorphisms and the risk of developing CRC in the Azerbaijani population. The study included 120 patients diagnosed with CRC and 125 healthy individuals. Peripheral blood samples were collected from all the subjects in EDTA tubes and DNA extraction was performed by salting out. Polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. While comparing without gender distinction no statistical correlation was found between the heterozygous TC (OR = 0.66; 95% CI = 0.37-1.15; p = 0.142), mutant CC (OR = 1.23; 95% CI = 0.62-2.45; p = 0.550), and mutant C (OR = 1.03; 95% CI = 0.72-1.49; p = 0.859) alleles of the miR-149 gene and the CT (OR = 1.23; 95% CI = 0.69-2.20; p = 0.485), mutant TT (OR = 1.29; 95% CI = 0.67-2.47; p = 0.452), and mutant T (OR = 1.17; 95% CI = 0.82-1.67; p = 0.388) alleles of the miR-196a2 gene and the risk of CRC. However, among women, miR-149 TC (OR = 0.43; 95% CI = 0.19-1.01; p = 0.048) correlated with a reduced risk of CRC, whereas miR-196a2 CT (OR = 2.77; 95% CI = 1.13-6.79; p = 0.025) correlated with an increased risk of CRC. Our findings indicated that miR-149 T>C (rs2292832) might play a protective role in the development of CRC in female patients, whereas the miR-196a2 (rs11614913) polymorphism is associated with an increased risk of CRC in women in the Azerbaijani population, highlighting the importance of gender dimorphism in cancer etiology.

Rhodopsin determinants for transducin activation: a gain-of-function approach.

Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein transducin. Non-helical portions of the C2- and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a "gain-of-function" mutagenesis to identify rhodopsin residues critical for transducin activation. Mutant opsins with residues 140-148 (C2-loop), 229-244 (C3-loop), or 310-320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with poly-Ala substitutions in the C2- and C3-loops formed the 500-nm absorbing pigments but failed to activate transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Significant ( approximately 50%) restoration of the rhodopsin/transducin coupling was achieved with re-introduction of residues Cys140/Lys141 and Arg147/Phe148 into the C2 template. The reverse substitutions of the C3-loop residues Thr229/Val230 and Ser240/Thr242/Thr243/Gln244 produced a pigment with a full capacity for transducin activation. The C4 template mutant was unable to bind 11-cis-retinal, and the presence of Asn310/Lys311 was required for correct folding of the protein. Subsequent mutagenesis of the C4-loop revealed the role of Phe313 and Met317. On the background of Asn310/Lys311, the inclusion of Phe313 and Met317 produced a mutant pigment with the potency of transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for transducin activation.

A GPR-protein interaction surface of Gi(alpha): implications for the mechanism of GDP-release inhibition.

Proteins containing G-protein regulatory (GPR) motifs represent a novel family of guanine nucleotide dissociation inhibitors (GDIs) for G(alpha) subunits from the Gi family. They selectively interact with the GDP-bound conformation of Gi(alpha) and transducin-alpha (Gt(alpha)), but not with Gs(alpha). A series of chimeric proteins between Gi(alpha)(1) and Gs(alpha) has been constructed to investigate GPR-contact sites on G(alpha) subunits and the mechanism of GPR-protein GDI activity. Analysis of the interaction of two GPR-proteins-AGS3GPR and Pcp2-with the chimeric G(alpha) subunits demonstrated that the GPR-Gi(alpha)(1) interface involves the Gi(alpha)(1) switch regions and Gi(alpha)(1)-144-151, a site within the helical domain. Residues within Gi(alpha)(1)-144-151 form conformation-sensitive contacts with switch III, and may directly interact with a GPR-protein or form a GPR-binding surface jointly with switch III. The helical domain site is critical to the ability of GPR-proteins to act as GDIs. Our data suggest that a mechanism of the GDI activity of GPR-proteins is different from that of GDIs for monomeric GTPases and from the GDI-like activity of G(betagamma) subunits. The GPR-proteins are likely to block a GDP-escape route on G(alpha) subunits.