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.
Primary hyperoxaluria 1 and renal Fanconi's syndrome are kidney diseases that share a common mechanism in which a key enzyme is mistargeted from peroxisomes to mitochondria. We have identified small molecules that correct the targeting defect and restore trafficking to peroxisomes. This proposal investigates the potential of using these molecules as a tool to understand the molecular basis of the disease and to serve as a strategy for therapeutics.
