Role of BPHL/homocysteine thiolactonase in hyperhomocysteinemia and disease

BPHL/同型半胱氨酸硫内酯酶在高同型半胱氨酸血症和疾病中的作用

基本信息

批准号：
9324453
负责人：
Judit Marsillach Lopez
金额：
$ 34.78万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-06 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9324453
关键词：
Address Albumins Alzheimer&apos s Disease Amino Acids Apolipoprotein A-I Architecture Arylesterase Atherosclerosis Autoimmune Diseases Binding Biological Biological Markers Blood Brain Cardiovascular Diseases Clinical Clinical Research Congenital Abnormality Data Detection Development Diet Disease Disease Marker Distress Dose Drug Metabolic Detoxication Enzymes Foundations Future Gene Expression Genetic Homocysteine Homocystine Human Hydrolysis Hyperhomocysteinemia Immunohistochemistry Inflammation Injection of therapeutic agent Kidney Kidney Failure Knock-out Knockout Mice Laboratories Lead Link Liver Lysine Mass Spectrum Analysis Mental disorders Methionine Methionine-tRNA Ligase Methods Modification Monitor Mus Nerve Degeneration Organ Pathology Physiological Plasma Plasma Proteins Play Population Prodrugs Proteins Protocols documentation RNA Recombinants Research Risk Factors Role Saline Seizures Study models Tissues Toxic effect Transgenic Mice Urine Valganciclovir Vitamin B Complex Vitamin B Deficiency Wild Type Mouse Work adduct aortic arch base biphenyl hydrolase-related protein bleomycin hydrolase disorder risk enzyme activity experience feeding histological stains improved in vivo insight loss of function nervous system disorder novel prevent protein biomarkers valacyclovir

项目摘要

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.

项目概要/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，包括心血管疾病、神经系统疾病和自身免疫性疾病。这些影响的机制没有被很好地理解。高血浆 Hcy 水平会导致高水平的同型半胱氨酸硫代内酯 (HCTL)，这是一种 Hcy 的有毒代谢物。 HCTL具有与蛋白质相互作用并修饰蛋白质的能力，从而产生蛋白质失活和功能丧失。这些修饰的生物学意义和后果尚不清楚已知，尽管它们可能引发上述疾病的发展。我们最近有表征了联苯水解酶样蛋白的非常有效的同型半胱氨酸硫代内酯酶活性 (BPHL)，之前研究了其在前药伐昔洛韦和缬更昔洛韦的生物活化中的作用。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)，之前已经描述过，BPHL 对 HCTL 具有更高的催化效率，表明 BPHL 在解毒 HCTL 中具有更显着的生理作用。拟议的研究旨在确定 BPHL 防止 HCTL 毒性的程度以及与高 Hcy/HCTL 水平相关的疾病的发展。此外，我们将证明在在高同型半胱氨酸血症的情况下，经 HCTL 修饰的血浆蛋白可以作为高同型半胱氨酸血症的生物标志物 HCTL 相关疾病风险。以下提出的具体目标利用了商业上可用的 BPHL 敲除 (BPHL-/-) 小鼠以及我们通过质谱 (MS) 鉴定蛋白质加合物的经验：目标 1 将检查 [无毒（子目标 1a）或有毒（子目标 1b）] 剂量注射 HCTL 对 BPHL-/- 和 BPHL+/+（野生型）小鼠与注射盐水的对照组进行比较。血浆蛋白白蛋白载脂蛋白 A-I 将分析赖氨酸残基上的 HCTL 加合物，并分析尿液中较高水平的使用 MS 分析分泌 HCTL。目标 2 将检查 BPHL-/- 小鼠调节以下因素影响的能力：由富含蛋氨酸的饮食引起的高同型半胱氨酸血症[伴有或不伴有 B 族维生素缺乏]。我们将检查小鼠组织的[早期]病理迹象，以及对基因表达的影响（子目标 2a）。我们还将鉴定目标 1（子目标 2b）中监测的相同蛋白质上的 HCTL 加合物。这些研究阐明了 BPHL 的新生理功能。由于 BPHL 的生理意义在我们最近的工作之前未知，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保护性同型半胱氨酸硫内酯酶。我们将还开发了基于 MS 的改进方法来监测 HCTL 加合物。由此得到的结果该提案将增加我们对高同型半胱氨酸血症与发展之间联系的理解疾病，并为检查 BPHL 遗传变异作为 HCTL-危险因素的影响提供基础相关疾病。