PROTEIN MODIFICATION BY NITRATION IN AGING RATS

衰老与血管疾病的增加有关。大量研究表明氧化应激导致血管老化功能受损，但其细胞分子机制尚未完全阐明。过氧亚硝酸盐和其他活性氮氧化物硝酸化蛋白质并改变其功能。肌浆网/内质网Ca ~（2+）ATP酶（SERCA）是调节Ca ~（2+）进入内质网和舒张血管平滑肌的重要因子，在衰老的骨骼肌和动脉粥样硬化的主动脉中被硝化失活。我们的假设是，硝化或氧化修饰的SERCA和其他蛋白质可能会导致血管功能障碍，在老化的主动脉。酪氨酸硝化会损害其他蛋白质的功能：另一种松弛调节剂（前列环素合酶），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）识别老化主动脉中的其他硝化蛋白。将使用Fisher 344大鼠（4月龄vs 25月龄）的主动脉。简言之，实验设计如下：目标#1（SERCA）：（1）通过测量主动脉环的等长张力进行功能研究，（2）通过测量45 Ca 2+摄取进行SERCA活性测定，（3）SERCA纯化和硝基酪氨酸（NO2-Tyr）检测，（4）SERCA的其他氧化修饰（蛋白质羰基、氧化甲硫氨酸）检测。硝基酪氨酸的检测将通过使用抗NO2-Tyr抗体的免疫学方法和通过HPLC-UV检测进行。其他修饰的检测将主要通过使用HPLC进行。目标二：（未知氧化蛋白）：（1）用抗NO2-Tyr抗体进行2D-凝胶和免疫印迹，（2）用抗NO2-Tyr抗体进行免疫沉淀，然后（a）用特异性抗体（例如Mn-SOD）进行免疫印迹，（B）通过氨基酸测序和/或质谱法鉴定蛋白质，（3）用抗NO2- Tyr抗体进行主动脉的免疫组织化学。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作为衰老进展的干预措施。

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.