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

使用酵母分析 PH1 相关丙氨酸：乙醛酸转氨酶 (AGT)

• 批准号: 7923388
• 负责人: Chandra L Tucker
• 金额: $ 33.05万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923388
• 关键词: Alanine; Alanine-glyoxylate aminotransferase; Alleles; Amino Acids; Binding; Biological Assay; Cells; Complement; Coupled; Data; Dihydrofolate Reductase; Dimerization; Disease; Enzyme Stability; Enzymes; Gel; Generic Drugs; Glyoxylates; Goals; Growth; Hot Spot; Human; In Vitro; Individual; Kidney Calculi; Knowledge; Libraries; Ligands; Maps; Mass Spectrum Analysis; Measurement; Measures; Methodology; Missense Mutation; Mitochondria; Molecular; Molecular Chaperones; Monitor; Mutation; Output; Patients; Peptides; Primary Hyperoxaluria; Process; Protein Conformation; Protein Deficiency; Proteins; Reporter; Research; Residual state; Sorting - Cell Movement; Structure; Techniques; Temperature; Therapeutic; Thermodynamics; Two-Hybrid System Techniques; Variant; Work; Yeasts; base; cell growth; cost; design; glyoxylate; high throughput screening; in vivo; insight; interest; mutant; novel; protein aggregation; protein folding; protein function; protein misfolding; public health relevance; research study; small molecule; yeast genetics; yeast two hybrid system

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.

描述（由申请人提供）：原发性高尿酸I型（PH1）是一种严重的肾结石疾病，由蛋白质丙氨酸：乙醛酸氨基转移酶（AGT）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余活性，因此它们代表了用于“矫正”治疗性治疗的有希望的候选物。一种这样的新兴治疗是使用药理学伴侣，其是能够通过稳定天然蛋白质构象来恢复蛋白质功能的小分子。虽然AGT的这种方法有很大的希望，但一个主要的障碍是缺乏可用于识别这些分子的简单的具有成本效益的测定。在拟议的研究中，我们将利用酵母的易处理遗传学来开发一种基于酵母的蛋白质稳定性测定方法，该方法使用简单的细胞生长作为输出。该稳定性测定和监测AGT催化活性的第二酵母互补测定将用于筛选可能拯救错误折叠的AGT变体的药理学分子伴侣。这些试验和已建立的酵母方法（双杂交）也将用于表征AGT的错误折叠变体。同时，我们将在体外研究PH1疾病突变的影响。体外研究将使用相对较新的质谱法SUPREX，可测定热力学稳定性值。这些实验的目的是为了更好地了解导致PH1疾病中蛋白质功能丧失的机制，并确定拯救错误折叠的AGT等位基因的小分子。更广泛地说，其目的是提供一种新的基于细胞的蛋白质错误折叠检测方法，可用于表征各种疾病相关蛋白质。公共卫生相关性这些研究将提供深入了解导致PH1疾病的分子机制，并可能确定新的化合物，以稳定AGT的突变版本。预期稳定性试验可广泛转移至其他蛋白质，并且不需要预先了解蛋白质功能。因此，拟议的工作有望用于治疗hyperoxlauria，但也有望与蛋白质错误折叠引起的许多其他疾病相关。