The accumulation of oxidatively modified proteins is an important hallmark of biological aging, often accompanied by conformational changes and/or loss of activity. However, in order to correlate specific protein modifications with functional consequences the exact nature of the modifications as well as their locations on the protein must be known. We have shown that the skeletal muscle sarcoplasmic reticulum (SR) Ca- ATPase shows an age-dependent loss of Cys on both isoforms, SERCA1 and SERCA2a. In addition, the SERCA2a selectively accumulates 3- nitrotyrosine on Tyr residues 294 and 295 at the boundary between luminal and transmembrane domain. Interestingly, only 3-nitrotyrosine formation but not Cys modification was functionally important. However, the SR Ca-ATPase from aged tissues was overall more sensitive to elevated temperature. In addition, our in vitro studies have shown that the SR Ca-ATPase is susceptible to redox modulation of activity via modification of specifically Cys-349. These findings lead to the two hypotheses which will be tested in this application. I. Age-dependent modification of Cys targets predominantly Cys residues which are not critical for activity but represent """"""""endogenous antioxidant Cys residues"""""""" that protect the protein from immediate inactivation. However, age- dependent Cys-modified Cys SR Ca-ATPase will be more sensitive to redox modulation of activity by various reactive oxygen and nitrogen species as well as S-nitrosothiols. II. Peroxynitrite is the species most likely responsible for SERCA2a nitration in vivo as it can cross membranes and has the potential to reach Tyr residues which are not accessible from the cytosol. These hypotheses will be tested in three Specific Aims. 1.) The identification of Cys residues of skeletal muscle SRCA1 and SERCA2a which suffer modification in vivo during aging; 2.) The characterization of the specific susceptibility of SERCA1 and SERCA2a from aged tissue to redox modulation of activity by reactive oxygen and nitrogen species and S-nitrosothiols; and 3.) The characterization of product patterns in the in vivo nitration of the SR Ca- ATPase by various defined nitrating systems/species under chemically controlled conditions to determine which of the species is actually able to nitrite the enzyme at Tyr at positions 294 and 295.
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