Analysis of PH1-associated alanine:glyoxylate aminotranferase (AGT) using yeast

描述(由申请人提供)：原发性高草酸尿症I型(PH1)是一种严重的肾结石疾病，由丙氨酸：乙醛转氨酶(AGT)蛋白缺乏引起。在许多患者中，AGT缺乏是由于错义突变降低了这种酶的稳定性，导致降解、错误定位或聚集。由于这些突变蛋白中的许多保留了残留活性，它们代表着有希望的“矫正”治疗候选药物。其中一种新兴的治疗方法是使用药理伴侣，这种小分子能够通过稳定天然蛋白质构象来恢复蛋白质功能。虽然这种方法对AGT有很大的前景，但一个主要的障碍是缺乏可用于识别这些分子的简单、经济有效的分析方法。在这项拟议的研究中，我们将利用酵母易处理的遗传学来开发一种基于酵母的蛋白质稳定性测试方法，该方法使用简单的细胞生长作为输出。这一稳定性试验和第二次监测AGT催化活性的酵母互补试验将被用于筛选可能拯救错误折叠的AGT变体的药理伴侣。这些检测，以及一种已建立的酵母菌方法，双杂交，也将被用于表征AGT的错误折叠变体。同时，我们将在体外检测PH1疾病突变的影响。体外研究将使用一种相对较新的质谱学方法SUPREX，它可以确定热力学稳定值。这些实验的目标是更好地了解导致PH1疾病蛋白质功能丧失的机制，并识别拯救错误折叠的AGT等位基因的小分子。更广泛地说，其目的是提供一种新的可推广的基于细胞的蛋白质错误折叠分析，可用于表征各种与疾病相关的蛋白质。公共卫生相关性这些研究将提供对导致PH1疾病的分子机制的洞察，并可能识别出稳定AGT突变版本的新化合物。稳定性分析有望广泛地转移到其他蛋白质上，并且不需要事先了解蛋白质的功能。因此，这项拟议的工作有望治疗高氧尿症，但也有望与蛋白质错误折叠引起的许多其他疾病相关。

DESCRIPTION (provided by applicant): Primary Hyperoxaluria type I (PH1) is a severe kidney stone disease caused by deficiency of the protein alanine: glyoxylate aminotransferase (AGT). In many patients, deficiency of AGT results from missense mutations that decrease the stability of this enzyme, causing degradation, mislocalization, or aggregation. Since many of these mutant proteins retain residual activity, they represent promising candidates for "corrective" therapeutic treatment. One such emerging treatment is the use of pharmacological chaperones, which are small molecules that are able to restore protein function by stabilizing a native protein conformation. While such an approach for AGT has great promise, a major impediment is the lack of simple cost-effective assays that can be used in identifying these molecules. In the proposed research, we will take advantage of the tractable genetics of yeast to develop a yeast-based assay for protein stability that uses simple cell growth as output. This stability assay, and a second yeast complementation assay that monitors AGT catalytic activity, will be used to screen for pharmacological chaperones that may rescue misfolded AGT variants. These assays, and a established yeast approach, two-hybrid, will also be used to characterize misfolded variants of AGT. In parallel, we will examine the effects of PH1 disease mutations in vitro. The in vitro studies will use a relatively new mass-spectrometry approach, SUPREX, that allows determination of thermodynamic stability values. The goals of these experiments are to better understand the mechanisms that result in loss of protein function in PH1 disease and to identify small molecules that rescue misfolded AGT alleles. More broadly, the aim is to deliver a novel generalizable cell-based assay for protein misfolding that can be used to characterize a variety of disease-associated proteins. PUBLIC HEALTH RELEVANCE These studies will provide insight into the molecular mechanisms leading to PH1 disease and may identify novel compounds that act to stabilize mutant versions of AGT. The stability assay is expected to be widely transferable to other proteins, and does not require prior knowledge of protein function. As such, the proposed work holds promise for the treatment of hyperoxlauria, but is also expected to have relevance for the numerous other diseases caused by protein misfolding.