Interaction between glomerular filtration barrier component actinin-4 and nephrosis-inducing factors

1.肾α-肌动蛋白-4的纯化及其与嘌呤霉素氨基核苷(PAN)的结合特性(参考文献1)我们建立了一种从哺乳动物肾脏中分离纯化肌动蛋白-4的有效方法。纯化的肌动蛋白-4具有已知的蛋白功能，并与PAN紧密结合，可诱发动物肾病。根据对肌动蛋白-4消化片段的分析结果，泛结合位点被定位在该蛋白的中心杆状光谱蛋白样重复序列中。在PAN肾病动物模型中，PAN可能通过与血影蛋白样重复序列结合而对Actinin-4的功能产生不利影响。2.散发性局灶节段性肾小球硬化(FGS)慢性肾功能衰竭患者ACTN4和podocin基因突变分析(参考文献2)我们从5例散发性FGS慢性肾功能衰竭患者的血液中提取mRNA，并制备cDNA。用ACTN-4和Podocin的特异性引物，进行聚合酶链式反应(PCR)。我们用直接测序的方法分析了ACTN4和Podocin基因的突变。在这两个基因中都没有发现导致氨基酸替换的突变。3.α-肌动蛋白-4与人蛋白多肽的相互作用(参考文献3)我们以人肌动蛋白-4蛋白样重复序列为诱饵，对人肾cDNA文库进行细菌双杂交筛选，鉴定人蛋白多肽为相互作用分子。我们通过体外亲和分析和培养细胞的免疫荧光研究进一步验证了这种相互作用。PAN对相互作用没有影响。原位杂交和免疫组织化学研究显示人蛋白在足细胞中有表达。这些结果表明，人蛋白是一种新的Actini-4类血影蛋白重复序列的结合伙伴。人素和PAN不太可能在肌动蛋白-4中竞争相同的结合表面。

1.Renal alpha-actinin-4 : Purification and puromycin aminonucleoside-(PAN)-binding property (Reference 1)We established an efficient purification method for actinin-4 from the mammalian kidney. The purified actinin-4 possessed the known function of the protein, and tightly bound to PAN that can induce nephrosis in animals. Based on the results of analysis of digested fragments of actinin-4, the PAN-binding site was mapped within the central rod spectrin-like repeats of the protein. It is possible that PAN has adverse effects on the function of actinin-4, by binding to the spectrin-like repeats, in the PAN nephrosis animal model.2.Analysis of mutations in alpha-actinin-4 (ACTN4) and podocin genes of patients with chronic renal failure due to sporadic focal segmental glomerulosclerosis (FGS) (Reference 2)We isolated mRNA from blood samples of five patients with chronic renal failure due to sporadic FGS, and then prepared cDNA. Using specific primers for ACTN-4 and podocin, polymareae chain reaction (PCR) was performed. We analyzed mutations in ACTN4 and podocin genes, by direct sequencing PCR products. Mutations resulting in amino acid substitutions were not found in both genes. This suggests that sporadic FSG is a heterogenous disease.3.Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide (Reference 3)We performed a bacterial two-hybrid screening of human kidney cDNA library using as a bait human actinin-4 spectrin-like repeats, and identified humanin peptide as an interacting molecule. We further verified this interaction by in vitro affinity assays and immunofluorescent studies of cultured cells. PAN had no effect on the interactions. In situ hybridization and Immunohistochemical studies showed the expression of humanin in podocytes. These results suggest that humanin is a novel binding partner of actini-4 spectrin-like repeats. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.