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.

R01DK081584

2008-09-01

2013-08-31