ABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.</p><p><b>METHODS</b>Constructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.</p><p><b>RESULTS</b>Tyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.</p><p><b>CONCLUSIONS</b>The activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.</p>
Subject(s)
Humans , Cell Line , Complement C3a , Metabolism , Complement C5a , Metabolism , Protein Binding , Protein Processing, Post-Translational , Receptor, Anaphylatoxin C5a , Metabolism , Receptors, Complement , Metabolism , Sulfotransferases , Genetics , Metabolism , Transfection , Tyrosine , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To identify the susceptibility genes of type 2 diabetes in Chinese Han population.</p><p><b>METHODS</b>Single nucleotide polymorphism (SNP) discovery, genotyping and haplotype construction were performed in 30 candidate genes. Case-control study were carried out in a population-based sample and confirmed by the transmission disequilibrium test (TDT) analysis in 77 trio pedigrees. The effects of the SNP rs5210 on gene expression were studied by reporter gene technique.</p><p><b>RESULTS</b>The case-control studies showed that several SNPs on KCNJ11 gene was associated with type 2 diabetes in Chinese Han population, in which the allele frequency of SNP rs5219, the genotype frequency of rs5210, rs2285676 and rs5219, and the frequency of haplotype GA combined of the rs5219 and rs5215 showed significant difference between these two groups (P < 0.05). In addition, TDT test also showed statistical significance on this haplotype GA (P < 0. 05). The reporter gene assay showed that the effect on gene expression was significantly different between two alleles of rs5210 (P < 0.05).</p><p><b>CONCLUSION</b>KCNJII gene is one of the susceptibility genes of type 2 diabetes in Chinese Han population.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Asian People , Genetics , Case-Control Studies , Diabetes Mellitus, Type 2 , Genetics , Genetic Predisposition to Disease , Genetic Testing , Genotype , Polymorphism, Single NucleotideABSTRACT
<p><b>OBJECTIVE</b>To locate the region responsible for nuclear localization of protein sAC.</p><p><b>METHODS</b>The eukaryotic expression vector of vairous sAC deletion mutants were transfected into Hela cells. The localization of each mutant was observed using confocal microscope.</p><p><b>RESULTS</b>For some mutants, the localization of sAC changed. Deletion of some region made it unable to locate in the nuclear.</p><p><b>CONCLUSION</b>It is possible to figure out that the nucleotide region (739-1038 and 1045-1261) take charge of nuclear localization of sAC.</p>