PROJECT SUMMARY/ABSTRACT Inborn errors of metabolism comprise a large class of genetic diseases involving disorders of cellular metabolism. Homocysteine is an intermediary metabolite derived from methionine that can either be recycled back to methionine, or shunted down the transsulfuration pathway by the action of cystathionine beta-synthase (CBS), an enzyme primarily expressed in the liver and kidney. Individuals with mutations in CBS have CBS deficiency, characterized by extreme elevations in plasma total homocysteine (tHcy) and phenotypes including increased incidence of thrombosis, osteoporosis, dislocated lenses, and mental retardation. In healthy adults, tHcy concentration in plasma ranges from 5 to 15 M, but untreated patients with CBS deficiency often have tHcy in excess of 200 M. Current treatment strategies involve dietary restriction and vitamin therapy, but these are only partially effective and do not work in all patients. Over 85% of the described mutations in CBS deficient patients are missense mutations in which a single incorrect amino acid is substituted into the CBS polypeptide. Over the past decade, our lab has developed six different humanized mouse models that each express a different patient-derived mutant human CBS protein using an inducible promoter expressed at high levels in the liver. In all of these models, liver CBS activity is greatly diminished (<5% of wild-type), and serum tHcy is elevated by at least 20-fold. However, the behavior of each mutant CBS protein is not identical, with mutations affecting protein stability, protein aggregation, enzymatic function, or a combination of all three. The decreased protein stability effects of the mutations are mediated via the ubiquitin/proteasome system. Amazingly, treatment of mutant-expressing mice with proteasome inhibitors (PIs) can functionally reverse the effects of a majority of the missense mutations, resulting in large increases in CBS activity in the liver, and in some cases lowering of tHcy to near wild-type levels. PIs are FDA approved drugs for the treatments of certain types of cancer, but have never been studied in the context of inborn errors of metabolism. Our data suggests that low-dose PI treatment may be able to restore sufficient CBS enzyme activity to be phenotypically beneficial, but not have the toxicities associated with the high levels used in cancer therapy. In addition, we have preliminary data that suggests the combination of PIs with other drugs that modulate the cellular proteostasis network, may be useful in increasing the effectiveness of PIs. Thus, the overall goal of the current proposal is to extend these studies and determine if PIs and proteostasis modulators are effective in restoring function to patient-derived CBS alleles. If successful, the experiments described here could lead to novel treatments of CBS deficiency and potentially other genetic diseases associated with missense mutations.

PROJECT NARRATIVE/RELEVANCE TO PUBLIC HEALTH Inborn errors of metabolism are individually rare but collectively are relatively common with an incidence of one in 1,500 individuals. In this proposal, we use cystathionine beta-synthase deficiency as a model to explore novel strategies to restore function to missense proteins by using drugs that alter the cellular folding environment. If this work is successful, it may develop into quickly translatable treatments for CBS deficiency and potentially a wide variety of other inborn errors of metabolism.