PROTEIN MODIFICATION BY NITRATION IN AGING RATS

衰老与血管疾病的增加有关。许多研究表明，氧化应激有助于血管老化的功能损伤，但其细胞分子机制尚不清楚。过氧亚硝酸盐和其他活性氮氧化物使蛋白质硝酸盐化并改变其功能。肌浆/内质网Ca2+ atp酶（SERCA）是Ca2+摄取到内质网储存和血管平滑肌松弛的重要调节剂，在老化的骨骼肌和动脉粥样硬化主动脉中被氮化和失活。我们的假设是SERCA等蛋白的硝化或氧化修饰可能导致老化主动脉的血管功能障碍。酪氨酸硝化损害其他蛋白质的功能：另一种松弛调节剂（前列腺环素合成酶），一种超氧化物清除剂（锰超氧化物歧化酶：Mn-SOD）和一种信号分子（磷脂酰肌醇3-激酶）在各种条件下被硝化。我的具体目标是：#1)确定SERCA是否被硝化或氧化修饰；# 2)鉴定老化主动脉中的其他硝化蛋白质。将使用Fisher 344大鼠（4月龄vs 25月龄）的主动脉。简而言之，实验设计如下：目的1 (SERCA):(1)通过主动脉环的等长张力测量进行功能研究，(2)通过45Ca2+摄取测量SERCA活性测定，(3)SERCA纯化和检测硝基酪氨酸（NO2-Tyr），(4)检测SERCA的其他氧化修饰（蛋白质羰基，氧化甲硫氨酸）。硝基酪氨酸的检测将采用抗n02 -Tyr抗体和高效液相色谱-紫外检测法进行。其他修饰的检测主要通过高效液相色谱法进行。目的2:（未知氧化蛋白）：(1)抗no2 -Tyr抗体的2d凝胶和免疫印迹，(2)抗n02 -Tyr抗体的免疫沉淀，随后(a)特异性抗体（如Mn- SOD）的免疫印迹，(b)通过氨基酸测序和/或质谱法鉴定蛋白质，(3)抗n02 -Tyr抗体的主动脉免疫组化。这些不同的方法将用于鉴定老化主动脉中的硝化蛋白。这些研究将让我们知道哪些蛋白质在衰老过程中被修改并导致血管功能障碍，并为我们提供使用抗氧化剂或氧化还原调节剂作为干预衰老进程的机会。

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.