Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
PLoS One. 2011;6(10):e26792. doi: 10.1371/journal.pone.0026792. Epub 2011 Oct 26.
ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect.
ADIPOQ 编码脂联素，是通过全基因组连锁分析鉴定的 2 型糖尿病（T2D）候选基因，有证据表明其蛋白功能在于增强胰岛素作用。为了研究导致泰国患者 T2D 的候选基因，我们鉴定了 10 种新的 ADIPOQ 变异，其中一些是非同义变异，仅在患者中观察到。为了研究这些非同义变异对脂联素结构和生化特性的影响，我们通过计算机建模对野生型和变异蛋白进行了结构分析，并进一步通过分子和生化研究对建模预测结构异常的变异进行了生化特性的表征。我们从研究中鉴定的变异（R55H、R112H 和 R131H）和之前的工作（G90S 和 R112C）中构建了包含野生型和变异 ADIPOQ cDNA 的重组质粒，并在培养的 HEK293T 细胞中转染瞬时表达和共表达，以研究它们的寡聚化、相互作用和分泌。我们发现新型 R55H 变异破坏了蛋白的多聚化，但对共表达的野生型蛋白没有影响，而新型 R131H 变异则影响蛋白的分泌，并以显性负性的方式影响共表达的野生型蛋白。R131H 变异能够从内质网运输到高尔基体、转高尔基网络和早期内体，但不能分泌。R131H 变异可能通过溶酶体系统降解，抑制其降解可使变异蛋白从分泌缺陷中恢复。我们已经表明，使用计算机建模预测氨基酸替代对脂联素寡聚化的影响是可能的。这也是首次报道 R131H 变异对蛋白分泌具有显性负效应，以及使用蛋白降解抑制剂作为携带脂联素变异、具有分泌缺陷的患者的治疗药物的可能性。
