Molecular Mechanism for Coronary Spastic Angina as a Transcriptional Factor-Related Disease

痉挛型心绞痛在日本人中比高加索人更常见。提示生殖道危险因素参与其发病机制。在此背景下，我们寻找与冠状动脉痉挛型心绞痛相关的内皮型一氧化氮合酶(ENOS)基因的多态性。我们发现eNOS基因外显子7的错义突变和eNOS基因5-侧翼区的单核苷酸多态性，外显子7的→C错义突变不影响eNOS的酶活性。而→C则使eNOS基因转录减少。为了阐明eNOS基因转录减少的分子机制，我们现在从HeLa细胞的核提取液中纯化了一种与突变等位基因特异结合的蛋白质。纯化的蛋白质与复制蛋白A1(RPA1)完全相同，RPA1是DNA修复所必需的单链DNA结合蛋白。复制和重组。在人脐静脉内皮细胞中，使用反义寡核苷酸抑制RPA1的表达恢复了由突变的启动子序列驱动的转录。反之，RPA1的过表达进一步降低了它的表达。RPA1在胎盘中也有类似的表达。携带T^-786&GT；→C突变的胎盘enos基因表达水平显著低于未携带该突变的胎盘。携带→C突变个体的血清亚硝酸盐/硝酸盐水平显著低于无突变个体，揭示了eNOS表达减弱的功能重要性。因此，RPA1在T^lt；-786&gt；→C突变相关的eNOS基因转录减少与冠状动脉疾病的发生相关中起着抑制蛋白的作用。

Cspastic angina is more frequently observed in Japanese than Caucasians. suggesting genctic risk factor is involved in its pathogenesis. In this context, we searched the polymorphisms in endothelial nitic oxide synthase (eNOS) gene, which are associated with coronary srastic angina. We found a missense mutation in exon 7 and a single nucletide polymorphism in the 5-flanking region of eNOS gene, T^<-786>→C Missense mutation in exon7 did not affect eNOS emzyme activity. but T^<-786>→C reduce eNOS gene transcription. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded-DNA binding protein essential for DNA repair. replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence. while conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta. and eNOS mRNA levels in placentas carrying the T^<-786>→C mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the T^<-786>→C mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the T^<-786>→C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.