Small Molecule Probes to Correct AGT Mistargeting in Primary Hyperoxaluria 1

 描述(申请人提供)：原发性高草酸尿I型(PH1)和肾脏Fanconi综合征是两种罕见的肾脏疾病，它们有一个共同的主题，即过氧化物体蛋白被错误地定位到线粒体。随着全基因组关联研究被诊断为其他肾脏疾病，这种蛋白质被错误定位为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退化的。PH1是一种常染色体隐性遗传病，由丙氨酸-乙氧基酸氨基转移酶(AGT/AGXT，蛋白质缩写)基因突变引起。AGT)。PH1的特点是不能有效地代谢乙醛，导致草酸钙在各种身体组织中积聚，特别是肾脏。除了可以通过透析暂时治疗的尿毒症外，患者还有其他并发症，如过量草酸盐引起的神经病变。服用吡哆醇(B6)，AGT的辅助因子，减轻了部分患者的一些症状；然而，缺乏适当的治疗，这种疾病通常是晚期的。虽然AGT的一些突变会导致蛋白质激活，但突变的一部分(三分之一的患者有一个等位基因有两个点突变，即P11L和G170R)会导致将功能性AGT从在人类(杂食动物)中活跃的过氧化体错误定位到在肉食动物中正常位置的线粒体。类似地，肾性Fanconi综合征是由于EHHADH编码的过氧化体双功能酶(PBE)错误定位于线粒体而引起的。PBE随后阻止线粒体脂肪酸氧化，导致一种常染色体显性疾病。我们的假设是，减少线粒体蛋白输入的小分子对于剖析AGT/PBE运输的分子机制具有重要意义，从长远来看，它可以作为一种治疗策略，将蛋白质从线粒体重新定位到过氧化物酶体。我们已经在酵母中完成了高通量的体内筛选，以寻找线粒体蛋白易位的衰减剂。强有力的初步数据支持，这些小分子探针减少了酵母和哺乳动物线粒体的输入。此外，小分子候选分子已被确定确实将AGT从线粒体重定向回过氧化体，并且这些探针对细胞具有很好的耐受性。因此，这些小分子在一定程度上抑制了线粒体蛋白的易位，而这种抑制对细胞无毒。这项建议的具体目的是：(1)阐明AGT在中国的具体转运途径 (2)研究小分子将AGT从线粒体重定向到过氧化体的机制，并确定小分子是否对AGT不同突变的患者有益；(3)研究PBE错误定位到线粒体的转运途径。这项研究与公共健康相关，因为有可能开发新的策略来了解和治疗肾脏疾病。

 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.