PROJECT SUMMARY/ABSTRACT Elevated levels of the amino acid homocysteine (Hcy) are a risk factor for a wide variety of disorders, from cardiovascular disease to neurological and autoimmune disorders. The mechanisms underlying these effects are not well understood. High plasma levels of Hcy lead to high levels of homocysteine thiolactone (HCTL), a toxic metabolite of Hcy. HCTL has the ability to interact with and modify proteins, resulting in protein inactivation and loss of function. The biological significance and consequence of these modifications is not well known, although they may trigger the development of the diseases noted above. We have recently characterized a very efficient homocysteine thiolactonase activity of the enzyme biphenyl hydrolase-like protein (BPHL), previously studied for its role in bioactivating the prodrugs valacyclovir and valganciclovir. Although two other enzymes with homocysteine thiolactonase activity, paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh), had previously been described, BPHL has a much higher catalytic efficiency for HCTL, indicating a more significant physiological role for BPHL in detoxifying HCTL. The proposed research aims to establish the extent to which BPHL protects against HCTL toxicity and the development of diseases associated with high Hcy/HCTL levels. In addition, we will demonstrate that in conditions of hyperhomocysteinemia, plasma proteins that are modified by HCTL can serve as biomarkers of HCTL-associated disease risk. The following proposed specific aims make use of the commercially-available BPHL knockout (BPHL-/-) mouse and our experience in identifying protein adducts by mass spectrometry (MS): Aim 1 will examine the effects of a [non-toxic (sub-Aim 1a) or toxic (sub-Aim 1b)] dose injection of HCTL on BPHL-/- and BPHL+/+ (wild-type) mice compared with controls injected with saline. The plasma proteins albumin and apolipoprotein A-I will be analyzed for HCTL adducts on lysine residues, and the urine for higher levels of secreted HCTL using MS analyses. Aim 2 will examine the ability of BPHL-/- mice to modulate the effects of hyperhomocysteinemia generated by diets high in methionine [with or without deficiency in B vitamins]. We will examine mouse tissues for [early] signs of pathology, together with effects on gene expression (sub-Aim 2a). We will also identify HCTL adducts on the same proteins monitored in Aim 1 (sub-Aim 2b). These studies elucidate a novel physiological function of BPHL. Since the physiological significance of BPHL was unknown prior to our recent work, the results obtained from the proposed research have the potential to define the role of BPHL as that of a physiologically significant, protective homocysteine thiolactonase. We will also develop improved MS-based methods for monitoring HCTL adducts. The results obtained from this proposal will increase our understanding of the link between hyperhomocysteinemia and development of disease, and provide a basis for examining the effects of genetic variability in BPHL as a risk factor for HCTL- associated diseases.