Role of BPHL/homocysteine thiolactonase in hyperhomocysteinemia and disease

项目摘要/摘要 氨基酸同型半胱氨酸(Hcy)水平升高是多种疾病的危险因素，从 从心血管疾病到神经和自身免疫性疾病。这些效应背后的机制 都没有被很好地理解。血浆同型半胱氨酸水平高会导致同型半胱氨酸硫内酯(HCTL)水平升高 同型半胱氨酸的有毒代谢产物。HCTL具有与蛋白质相互作用和修饰蛋白质的能力，从而产生蛋白质 失活和丧失功能。这些修饰的生物学意义和后果并不是很好 已知的，尽管它们可能引发上述疾病的发展。我们最近做了 表征了一种非常高效的同型半胱氨酸硫代内酯酶活性的联苯水解酶样蛋白 (BPHL)，先前因其在生物激活前药物valacylovir和valganciclovir中的作用而被研究。虽然 另外两种具有同型半胱氨酸硫代内酯酶活性的酶，对氧磷酶-1(PON1)和博莱霉素水解酶 (BLMH)，BPHL对HCTL具有更高的催化效率，表明 Bphl在解毒方面有更显著的生理作用。 拟议的研究旨在确定BPHL对HCTL毒性的保护程度，以及 与高Hcy/HCTL水平相关的疾病的发生。此外，我们还将在 高同型半胱氨酸血症条件下，经HCTL修饰的血浆蛋白可作为 与HCTL相关的疾病风险。以下建议的具体目标利用了商业上可获得的 BPHL基因敲除(BPHL-/-)小鼠和我们用质谱仪(MS)鉴定蛋白质加合物的经验： 目标1将检查注射[无毒(次级目标1a)或有毒(次级目标1b)]剂量的HCTL对 BPHL-/-和BPHL+/+(野生型)小鼠与注射生理盐水的对照组进行比较。血浆蛋白白蛋白 将分析载脂蛋白A-I在赖氨酸残基上的HCTL加合物，以及尿液中较高水平的 用MS分析分泌的HCTL。目的2将检测BPHL-/-小鼠调节 高同型半胱氨酸血症是由于饮食中蛋氨酸含量高[是否伴有维生素B缺乏]引起的。我们会 检查小鼠组织的[早期]病理迹象，以及对基因表达的影响(分目标2a)。 我们还将确定目标1(亚目标2b)中监测到的相同蛋白质上的HCTL加合物。 这些研究阐明了BPHL的一种新的生理功能。既然BPHL的生理学意义 在我们最近的工作之前是未知的，从拟议的研究中获得的结果有可能 将BPHL的作用定义为具有生理意义的、具有保护性的同型半胱氨酸硫代内酯酶。我们会 也开发改进的以MS为基础的监测HCTL加合物的方法。从这个过程中得到的结果 该提案将增加我们对高同型半胱氨酸血症与糖尿病发展之间的联系的了解 疾病，并为研究BPHL的遗传变异性作为HCTL的危险因素的影响提供了基础。 相关疾病。

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.