Small Molecule Probes to Correct AGT Mistargeting in Primary Hyperoxaluria 1

用于纠正原发性高草酸尿症中 AGT 误定位的小分子探针 1

基本信息

批准号：
9130819
负责人：
Carla M Koehler
金额：
$ 23.1万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-21 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9130819
关键词：
Alanine-glyoxylate aminotransferase Alleles Area Attenuated Back Biogenesis C-terminal Calcium Oxalate Cell Line Cell model Cells Chemicals Chinese Hamster Ovary Cell Chloride Ion Chlorides Code Collaborations Collection Cultured Cells Cytosol Data Defect Development Dialysis patients Disease Enzymes Event FDA approved Fanconi Syndrome Fibroblasts Future Genes Genetic Polymorphism Genetic Screening Glutamates Glycine Goals Health Human Kidney Kidney Calculi Kidney Diseases Lead Left Letters Life Expectancy Location Lysine Mitochondria Mitochondrial Proteins Modeling Molecular Mutation N-terminal Neuropathy Oxalates Pathway interactions Patients Peptide Hydrolases Point Mutation Positioning Attribute Primary Hyperoxaluria Process Protein Import Protein translocation Proteins Public Health Publications Research Supplementation Symptoms Testing Therapeutic Tissues Uremia Vitamin B6 Yeasts base cofactor disease diagnosis disease-causing mutation exome sequencing expectation fatty acid oxidation genome wide association study glyoxylate in vivo induced pluripotent stem cell insight interest liver transplantation mitochondrial processing peptidase mutant novel therapeutic intervention patient subsets peroxisome protein activation protein transport reconstitution small molecule success tool trafficking yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Primary hyperoxaluria type I (PH1) and renal Fanconi's syndrome are two rare kidney diseases that share a common theme in which a peroxisomal protein is mistargeted to mitochondria. As additional kidney diseases are diagnosed with genome-wide association studies, this theme in which proteins are mistargeted to mitochondria is likely to become more common, because mitochondrial targeting sequences are degenerate. PH1 is an autosomal recessive disease caused by mutations in the gene coding for alanine-glyoxylate aminotransferase (AGT/AGXT, protein abbrev. AGT). PH1 is marked by an inability to efficiently metabolize glyoxylate, leading to the accumulation of calcium oxalate in various bodily tissues, especially the kidney. In addition to uremia, which can be transiently treated by dialysis, patients have other complications such as neuropathy from excess oxalates. Administration of pyridoxine (B6), a cofactor of AGT, has alleviated some symptoms in a subset of patients; however, adequate treatments are lacking and the disease is typically terminal. Whereas some mutations in AGT result in protein activation, a subset of mutations (one-third of patients have an allele with 2 point mutations, P11L and G170R) results in mistargeting of functional AGT from peroxisomes, where it is active in humans (omnivores), to mitochondria, which is the normal location in carnivores. Similarly, renal Fanconi's syndrome is caused by mistargeting of the peroxisomal bifunctional enzyme (PBE, coded by EHHADH) to mitochondria. PBE subsequently blocks mitochondrial fatty acid oxidation, resulting in an autosomal dominant disease. Our hypothesis is that small molecules that attenuate mitochondrial protein import are significant for dissecting the molecular mechanisms in AGT/PBE trafficking and, long-term, as a therapeutic strategy to retarget the protein from mitochondria back to peroxisomes. We have completed a high throughput in vivo screen in yeast to find attenuators of mitochondrial protein translocation. Strong preliminary data supports that these small molecule probes attenuate import in both yeast and mammalian mitochondria. Moreover, small molecule candidates have been identified that indeed retarget AGT from mitochondria back to peroxisomes and these probes are well tolerated by cells. Thus, the small molecules partially inhibit mitochondrial protein translocation at a level that is not toxic to cels. The specific aims of this proposal are: (1) to elucidate the specific trafficking pathway of AGT in cells; (2) to characterize the mechanism by which the small molecules retarget AGT from mitochondria to peroxisomes and to determine if the small molecules may be beneficial for patients with different mutations in AGT; and (3) to study the trafficking pathway of PBE mistargeting to mitochondria. This study is relevant to public health because of the potential development of new strategies to understand and treat kidney diseases.

描述（由申请人提供）：原发性高草酸尿症 I 型 (PH1) 和肾范可尼综合征是两种罕见的肾脏疾病，它们有一个共同的主题，即过氧化物酶体蛋白被错误定位到线粒体，同时通过全基因组关联研究诊断出其他肾脏疾病。，这种蛋白质错误定位到线粒体的主题可能会变得更加常见，因为线粒体靶向序列是简并的，PH1 是一种由基因编码突变引起的常染色体隐性遗传疾病。丙氨酸乙醛酸转氨酶（AGT/AGXT，蛋白质缩写。AGT）的特点是无法有效代谢乙醛酸，导致草酸钙在身体各组织中积聚，尤其是肾脏，这可能导致尿毒症。如果暂时通过透析进行治疗，患者会出现其他并发症，例如因服用过量草酸盐（B6）（一种辅助因子）而引起的神经病变。 AGT 缓解了一部分患者的一些症状；然而，由于缺乏足够的治疗，该疾病通常已处于晚期，而 AGT 的一些突变会导致蛋白质激活，这是突变的一个子集（三分之一的患者具有 2 的等位基因）。点突变（P11L 和 G170R）导致功能性 AGT 从过氧化物酶体（在人类（杂食动物）中活跃）错误定位到线粒体（类似地，在肉食动物中，线粒体是肾脏的正常位置）。范科尼综合征是由过氧化物酶体双功能酶（PBE，由 EHHADH 编码）错误定位至线粒体引起的，PBE 随后会阻止线粒体脂肪酸氧化，从而导致常染色体显性遗传疾病。我们的假设是，减弱线粒体蛋白输入的小分子对线粒体具有重要意义。剖析 AGT/PBE 运输的分子机制，并作为将蛋白质从线粒体重新定位回过氧化物酶体的长期治疗策略，我们已经完成了一项高水平的研究。强有力的初步数据支持这些小分子探针可以减弱酵母和哺乳动物线粒体中的输入，此外，已经鉴定出确实将 AGT 从线粒体重新定位回过氧化物酶体的小分子候选物。这些探针对细胞具有良好的耐受性，因此，小分子在对细胞无毒的水平上部分抑制线粒体蛋白易位。 AGT 的具体转运途径 (2) 表征小分子将 AGT 从线粒体重新靶向到过氧化物酶体的机制，并确定小分子是否可能对 AGT 不同突变的患者有益；(3) 研究 PBE 错误靶向线粒体的运输途径；这项研究与公共卫生相关，因为它有可能开发出了解和治疗肾脏疾病的新策略。