Mutant Laminin Chains and Nephrotic Syndrome

描述（由申请人提供）：层粘连蛋白2（LAMB 2）是层粘连蛋白521的一种组分，层粘连蛋白521是肾小球基底膜（GBM）的主要层粘连蛋白三聚体。LAMB 2中的突变导致皮尔逊综合征，其特征在于具有严重的眼部和神经缺陷的先天性肾病综合征。相反，大多数错义LAMB 2突变，如R246 Q，导致孤立的先天性肾病综合征。为了研究错义突变引起蛋白尿的机制，我们已经产生了转基因小鼠，其中R246 Q突变体β 2在足细胞中以不同水平表达，并取代GBM中的正常β 2。我们的初步数据表明，这些小鼠表现出蛋白尿，可以通过增加突变体R246 Q-LAMB 2沉积到GBM中来减轻。此外，在体外，与野生型相比，突变LAMB 2蛋白的分泌受到严重抑制。因此，我们假设致病性LAMB 2点突变导致蛋白质错误折叠，ER应激和分泌缺陷在体外和体内。化学分子伴侣，如牛磺脱氧胆酸（TUDCA），是低分子量的化合物，可以稳定蛋白质构象和拯救运输缺陷的错误折叠的蛋白质。我们已经表明，表达突变体的细胞的TUDCA处理显著增加了突变体LAMB 2蛋白向培养基中的分泌。因此，我们提出，化学分子伴侣可以改善突变LAMB 2蛋白的折叠，减少ER应激，并改善突变层粘连蛋白三聚体分泌到GBM中，从而减轻Lamb 2-/-; mutLAMB 2转基因小鼠的蛋白尿。为了验证这些假设，我们将： 1.检查LAMB 2点突变是否导致蛋白质错误折叠和ER应激 无论是在体外还是在体内。 2.确定化学分子伴侣是否可以改善LAMB 2点的分泌 突变体和减少ER应激。 3.研究化学分子伴侣是否改善突变层粘连蛋白的分泌（2和 减轻Lomb 2-/-; mutLAMB 2小鼠的蛋白尿。 这些研究将有助于我们了解先天性肾病综合征发病的分子机制，并在相关动物模型中分析化学分子伴侣治疗的可行性。我们的研究结果可能对治疗携带错义LAMB 2突变的先天性肾病综合征患者具有重要的治疗意义。 公共卫生相关性：先天性肾病综合征是一种重要的儿童肾脏疾病，可导致蛋白尿和进行性肾衰竭。我们正在动物模型中研究是否给予具有生物活性的分子伴侣可以改善蛋白尿。这一结果可能为肾性蛋白尿疾病提供一种新的治疗方案。

DESCRIPTION (provided by applicant): Laminin (2 (LAMB2) is a component of Laminin-521, the major laminin trimer of the kidney glomerular basement membrane (GBM). Null mutations in LAMB2 cause Pierson syndrome, characterized by congenital nephrotic syndrome with severe ocular and neurological defects. In contrast, most missense LAMB2 mutations, such as R246Q, cause isolated congenital nephrotic syndrome. To investigate the mechanisms whereby missense mutations cause proteinuria, we have generated transgenic mice in which R246Q mutant (2 is expressed in podocytes at different levels and replaces normal (2 in the GBM. Our preliminary data show that these mice exhibit proteinuria that can be attenuated by increased deposition of mutant R246Q-LAMB2 into the GBM. Moreover, in vitro, secretion of mutant LAMB2 proteins is severely inhibited compared to wild-type. Therefore, we hypothesize that pathogenic LAMB2 point mutations cause protein misfolding, ER stress, and secretion defects both in vitro and in vivo. Chemical chaperones, such as taurodeoxycholic acid (TUDCA), are low molecular weight compounds that can stabilize protein conformation and rescue trafficking-defective misfolded proteins. We have shown that TUDCA treatment of cells expressing the mutants significantly increases the secretion of mutant LAMB2 proteins into the medium. We propose, therefore, that chemical chaperones can improve the folding of mutant LAMB2 proteins, reduce ER stress, and improve the secretion of mutant laminin trimers into the GBM, thereby attenuating proteinuria in Lamb2-/-; mutLAMB2 transgenic mice. To test these hypotheses, we will: 1. Examine whether LAMB2 point mutations cause protein misfolding and ER stress both in vitro and in vivo. 2. Determine whether chemical chaperones can improve the secretion of LAMB2 point mutants and reduce ER stress in vitro. 3. Investigate whether chemical chaperones improve secretion of mutant laminin (2 and attenuate proteinuria in Lamb2-/-; mutLAMB2 mice. These studies will help us to understand the molecular mechanisms underlying the pathogenesis of congenital nephrotic syndrome and assay the feasibility for therapeutic use of chemical chaperones in a relevant animal model. Our results may have significant therapeutic implications for the treatment of congenital nephrotic syndrome in patients carrying missense LAMB2 mutations. PUBLIC HEALTH RELEVANCE: Congenital nephrotic syndrome is an important childhood kidney disease which causes proteinuria and progressive renal failure. We are studying whether the administration of pharmacologically active chaperones can ameliorate proteinuria in an animal model. The results may indicate a new treatment scheme for renal proteinuric diseases.