BIOLOGY OF PROTEOLYTIC DERIVATIVES OF LP(A)

LP(A) 蛋白水解衍生物的生物学

• 批准号: 6286244
• 负责人: Angelo M Scanu
• 金额: $ 33.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6286244
• 关键词: apolipoproteins; atherosclerotic plaque; blood lipoprotein metabolism; blood tests; carotid artery; disease /disorder model; embryo /fetus tissue /cell culture; genetic transduction; human subject; human tissue; immunocytochemistry; laboratory mouse; metalloendopeptidases; molecular pathology; plasmapheresis; protein localization; protein structure function; proteolysis; site directed mutagenesis; urinalysis

DESCRIPTION: The presence of immunoreactive fragments of apolipoprotein(a), apo(a), have been shown in the plasma, urine anc atheromas of human subjects. Moreover, derivatives of lipoprotein(a), Lp(a), and apo(a) can be generated in vitrc by the action of enzymes of the elastase and metalloproteinase (MMP) families. The goal of our proposed studies is to shed light on these products, in terms of their potential participation in the cardiovascular pathogenicity ol Lp(a). In particular, we wish to test the hypothesis that enzymes of the MMP family shown in vitro to cleave Lp(a; in the hot spot of the linker region between kringle IV-4 and IV-5 of apo(a), generate at tissue sites, two mair derivatives. One, miniLp(a), a particle that has as a protein moiety apoB 100 linked to P2, the C-terminal fragmen of apo(a) able to bind to members of the vascular matrix. The other, F1 representing the N- terminal domain of spo(a) containing the kringle IV-2 repeats and, lacking matrix binding function. A portion of F1 once releasec from apo(a) at tissue sites, would return to the plasma and then rapidly excreted into the urine. In turn. F2. eithe free or a member of a miniLp(a), would be preferentially retained at the sites of formation, preferentially in the sub endotheial intima where it undergoes atherogenic changes. This hypothesis wifi be tested by four relatec approaches: 1) structural, functional and immunological studies on the properties of apo(a) in order to define its various domains, and the basis for the cleavage specificities by MMPs; 2) on the premise that the linker 4 is thc one most susceptible to MMP cleavage, express in vitro apo(a) species having linker 4 mutated to be resistant t MMP cleavage, and then compare the in vitro and in vivo properties of these mutants with those of their wild-type counterpart; 3) introduce via adenovirus technology in apoE mice and crosses between apoE-/- and human apoB100 transgenics in different stages of atherogenesis, either Fl, P2, wild-type or linker 4-mutated apo(a) anc assess the localization of these products in unaffected and lesion areas along with measurements of MMI activities; 4) study surgical segments of human carotid arteries with either stable or unstable plaques to determine using immunochemical and chemical methods, the comparative localization of apo(a) and fragments in these tw types of plaques and also determine whether the fragments resemble those generated in vitro from the digestion o Lp(a)/apo(a) with MMPs. The combined in vitro and in vivo studies are expected to improve our knowledge oi the properties of the various domains of apo(a) and shed light on the potential role that MMP-mediated proteoIysis may play in the atherogenicity of Lp(a)/apo(a) in the context of the general inflammatory theory of atherosclerosis.

描述：存在载脂蛋白（a）的免疫反应性片段， 载脂蛋白（a）已在人类受试者的血浆、尿液和动脉粥样硬化中显示。 此外，脂蛋白（a）、脂蛋白（a）和载脂蛋白（a）的衍生物可以在 通过弹性蛋白酶和金属蛋白酶（MMP）的酶的作用， 家庭我们提出的研究的目标是阐明这些产品， 就其在心血管致病性中的潜在参与而言， ol Lp（a）.特别地，我们希望测试MMP的酶 在体外显示出在接头区的热点中切割Lp（a; 在apo（a）的kringle IV-4和IV-5之间，在组织部位产生，两个主要的 衍生物.一种是miniLp（a），一种含有apoB 100蛋白质部分的颗粒 与P2连接的apo（a）的C末端片段能够结合蛋白质的成员 血管基质F_1代表spo（a）的N-末端结构域; 含有Kringle IV-2重复序列，缺乏基质结合功能。一 F1的一部分一旦在组织部位从apo（a）中释放出来，就会回到组织中。 血浆中，然后迅速排泄到尿液中。依次F2。无论是自由的还是 miniLp（a）的成员，将优先保留在 形成，优先在内膜下，在那里它经历 致动脉粥样硬化的变化这一假设可以通过四种相关的方法来检验： 1)载脂蛋白（a）的结构、功能和免疫学研究 以确定其不同的结构域，以及切割的基础 2）在连接子4是MMPs的最大特异性的前提下， 对MMP切割敏感，体外表达具有接头4 apo（a）种类 突变为抗tMMP切割，然后比较体外和体内 这些突变体与野生型对应物的体内特性; 3） 通过腺病毒技术导入apoE小鼠以及apoE-/- 和人apoB 100转基因体在动脉粥样硬化形成的不同阶段，F1， P2，野生型或接头4突变的载脂蛋白（a）和评估这些蛋白的定位。 沿着MMI测量的未受影响和病变区域的产品 活动; 4）研究人类颈动脉的手术段， 稳定或不稳定斑块，以确定使用免疫化学和化学 方法对这两种类型的载脂蛋白（a）及其片段进行比较定位， 并确定这些片段是否类似于 用MMP消化Lp（a）/apo（a）。结合体外和体内 体内研究有望提高我们对这些特性的认识。 载脂蛋白（a）的各个领域，并阐明其潜在作用， MMP介导的蛋白水解可能在Lp（a）/apo（a）致动脉粥样硬化中起作用。 动脉粥样硬化的一般炎症理论的背景下。