PROTEIN MODIFICATION BY NITRATION IN AGING RATS

Boston Medical Center, Boston MA

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Abstract

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.