PROTEIN MODIFICATION BY NITRATION IN AGING RATS

衰老大鼠中蛋白质的硝化修饰

• 批准号: 6287935
• 负责人: Reiko Matsui
• 金额: $ 8.01万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6287935
• 关键词: aging; aorta; calcium transporting ATPase; endoplasmic reticulum; gel electrophoresis; immunoprecipitation; laboratory rat; mass spectrometry; nitrates; oxidation; sarcoplasmic reticulum; striated muscles

Aging is associated with increased vascular disease. A number of studies suggest that oxidative stress contributes to functional impairment of aging vessels, but the cellular molecular mechanisms are not well elucidated. Peroxynitrite and other reactive nitrogen oxides nitrate proteins and modify their function. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), an important regulator of Ca2+ uptake into ER stores and relaxation of vascular smooth muscle, is nitrated and inactivated in aging skeletal muscle and in atherosclerotic aorta. Our hypothesis is that nitration or oxidative modification of SERCA and other proteins may result in vascular dysfunction in aging aorta. Tyrosine nitration impairs function of other proteins: another regulator for relaxation (prostacyclin synthase), a superoxide scavenger (manganese superoxide dismutase:Mn-SOD), and a signaling molecule (phosphatidylinositol 3-kinase) are nitrated in various conditions. My specific aims are: #1) To determine if SERCA is modified by nitration or oxidation, # 2) To identify other nitrated proteins in aging aorta. Aortas from Fisher 344 rats (4 month old vs 25 month old) will be used. Briefly, experiments are designed as follows: Aim #1 (SERCA): (1) functional studies by isometric tension measurement of aortic rings, (2) SERCA activity assay by 45Ca2+ uptake measurement, (3) SERCA purification and detection of nitrotyrosine (NO2-Tyr), (4) detection of other oxidative modifications (protein carbonyl, oxidative methionine) of SERCA. Detection of nitrotyrosine will be done by immunological methods with anti-N02 -Tyr antibodies and by HPLC-UV detection. Detection of other modifications will be mainly done by using HPLC. Aim #2: (Unknown oxidized proteins): (1) 2D-gel and immunoblot with anti-NO2-Tyr antibodies, (2) immunoprecipitation with anti-N02-Tyr antibodies followed by (a) immunoblot with specific antibodies (e.g. Mn- SOD), (b) identification of proteins by amino acid sequencing and/or mass spectrometry, (3) immunohistochemistry of aorta with anti-N02- Tyr antibodies. These different approaches will be used to identify nitrated protein(s) in aging aorta. These studies will let us know which protein(s) are modified and cause vascular dysfunction during aging, and give us opportunities to use anti- oxidants or redox modulators as an intervention to progression of aging.

衰老与血管疾病的增加有关。大量研究表明，氧化应激参与了衰老血管的功能损害，但其细胞分子机制尚不清楚。过氧亚硝酸盐和其他反应性氮氧化物可抑制硝酸盐蛋白质并改变其功能。肌浆/内质网钙ATPase(SERCA)是内质网钙摄取和血管平滑肌松弛的重要调节因子，在衰老的骨骼肌和动脉粥样硬化的动脉中被硝化和失活。我们的假设是，SERCA和其他蛋白质的硝化或氧化修饰可能导致衰老的主动脉血管功能障碍。酪氨酸硝化会损害其他蛋白质的功能：另一种松弛调节因子(前列环素合成酶)、超氧化物清除剂(锰超氧化物歧化酶：Mn-SOD)和信号分子(磷脂酰肌醇3-激酶)在不同的条件下被硝化。我的具体目标是：#1)确定SERCA是否被硝化或氧化修饰，#2)识别老化的主动脉中的其他硝化蛋白。将使用Fisher 344只大鼠(4月龄和25月龄)的主动脉。实验设计如下：目的1(SERCA)：(1)主动脉环等长张力功能研究；(2)SERCA活性测定；(3)SERCA纯化及硝基酪氨酸(NO2-Tyr)的检测；(4)SERCA其他氧化修饰(蛋白质羰基、氧化蛋氨酸)的检测。硝基酪氨酸的检测将通过使用抗N02-Tyr抗体的免疫学方法和高效液相-紫外检测来进行。其他修饰的检测将主要通过高效液相色谱法进行。目的#2：(未知氧化蛋白)：(1)二维凝胶和抗NO2-Tyr抗体免疫印迹；(2)抗N02-Tyr抗体免疫沉淀；(A)特异性抗体免疫印迹(如：Mn-SOD)；(B)氨基酸序列和/或质谱学鉴定蛋白质；(3)抗N02-Tyr抗体免疫组织化学。这些不同的方法将被用来识别老化的主动脉中的硝化蛋白(S)。这些研究将让我们知道哪些蛋白质(S)在衰老过程中被修饰并导致血管功能障碍，并使我们有机会使用抗氧化剂或氧化还原调节剂来干预衰老的进展。

项目成果

Attenuated cardiovascular hypertrophy and oxidant generation in response to angiotensin II infusion in glutaredoxin-1 knockout mice.

• DOI: 10.1016/j.freeradbiomed.2010.07.005
• 发表时间: 2010-10-15
• 期刊: FREE RADICAL BIOLOGY AND MEDICINE
• 影响因子: 7.4
• 作者: Bachschmid, Markus M.;Xu, Shanqin;Maitland-Toolan, Karlene A.;Ho, Ye-Shih;Cohen, Richard A.;Matsui, Reiko
• 通讯作者: Matsui, Reiko