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

1.肾脏α -actin -4：纯化及嘌呤霉素氨基核苷（PAN）结合特性（文献1）我们建立了从哺乳动物肾脏中纯化α -actin -4的高效方法。纯化得到的肌动蛋白4具有已知的肌动蛋白功能，并能与可诱导动物肾病的PAN紧密结合。根据对actiin -4酶切片段的分析结果，pan结合位点被定位在该蛋白的中心杆谱样重复序列中。在PAN肾病动物模型中，PAN可能通过与谱蛋白样重复序列结合而对肌动蛋白-4的功能产生不利影响。散发性局灶节段性肾小球硬化（FGS）慢性肾衰竭患者α -肌动蛋白-4 （ACTN4）和podocin基因突变分析（文献2）我们从5例散发性局灶节段性肾小球硬化（FGS）慢性肾衰竭患者的血液样本中分离mRNA，制备cDNA。采用acn -4和podocin特异性引物，进行聚合酶链反应（PCR）。我们通过直接测序PCR产物分析了ACTN4和podocin基因的突变。在这两个基因中没有发现导致氨基酸替换的突变。这表明散发性FSG是一种异质性疾病。本文以人actinin-4 spectrin-like repeats为诱饵，对人肾脏cDNA文库进行细菌双杂交筛选，鉴定出人蛋白肽是一个相互作用分子。我们通过体外亲和实验和培养细胞的免疫荧光研究进一步证实了这种相互作用。PAN对相互作用没有影响。原位杂交和免疫组化研究显示人蛋白在足细胞中表达。这些结果表明humanin是actni -4 spectrin样重复序列的一个新的结合伙伴。人源蛋白和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.