Small Molecule Probes to Correct AGT Mistargeting in Primary Hyperoxaluria 1

 描述（由申请人提供）：原发性高尿酸I型（PH 1）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错误定位于线粒体。随着全基因组关联研究诊断出更多的肾脏疾病，蛋白质错误定位到线粒体的这一主题可能变得更加常见，因为线粒体靶向序列是简并的。PH 1是一种常染色体隐性遗传疾病，由编码丙氨酸-乙醛酸氨基转移酶（AGT/AGXT，蛋白质焦v. AGT）的基因突变引起。PH 1的特点是不能有效地代谢乙醛酸，导致草酸钙在各种身体组织，特别是肾脏中的积累。除了可以通过透析暂时治疗的尿毒症之外，患者还具有其他并发症，例如过量尿酸盐引起的神经病变。吡哆醇（B6），AGT的辅因子，管理，减轻了一些症状的患者的子集，但是，缺乏足够的治疗，疾病通常是终端。尽管AGT中的一些突变导致蛋白质活化，但突变的子集（三分之一的患者具有具有两个点突变P11 L和G170 R的等位基因）导致功能性AGT从过氧化物酶体（其在人类（杂食动物）中具有活性）到线粒体（其是食肉动物中的正常位置）的错误定位。类似地，肾范可尼综合征是由过氧化物酶体双功能酶（PBE，由EHHADH编码）错误定位于线粒体引起的。PBE随后阻断线粒体脂肪酸氧化，导致常染色体显性疾病。 我们的假设是，减少线粒体蛋白质输入的小分子对于解剖AGT/PBE运输的分子机制是重要的，并且长期而言，作为将蛋白质从线粒体重新靶向至过氧化物酶体的治疗策略。我们已经完成了一个高通量在酵母体内筛选，以找到线粒体蛋白质易位的衰减剂。强有力的初步数据支持这些小分子探针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从线粒体重新靶向回到过氧化物酶体的小分子候选物，并且这些探针被细胞很好地耐受。因此，小分子部分抑制线粒体蛋白质易位在一个水平，这是没有毒性的大肠杆菌。该建议的具体目标是：（1）阐明AGT在体内的特定转运途径， 单元格;（2）研究小分子药物将AGT从线粒体转移到过氧化物酶体的机制，并确定小分子药物是否对AGT不同突变的患者有益;（3）研究PBE错误转移到线粒体的运输途径。这项研究与公共卫生有关，因为它可能会开发出了解和治疗肾脏疾病的新策略。

 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.