Protein tyrosine nitration in rat brain is associated with raft proteins, flotillin-1 and alpha-tubulin: effect of biological aging.
Protein 3-nitrotyrosine (3-NY) immunoreactivity of rat brain homogenate was localized to a ca. 50 kDa protein band by western blot (WB) analysis. The nitrated proteins were localized to the raft fraction obtained by centrifugation of the homogenate in a sucrose density gradient, which contained specific raft markers such as flotillin-1 and caveolin-1. Purification of the nitrated raft proteins either by a combination of reversed-phase high-performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or by immunoprecipitation (IP) with protein- and modification-specific antibodies coupled to WB and HPLC-electrospray ionization-tandem mass spectrometry (ESI--MS/MS) analysis allowed us to identify two proteins modified by 3-NY: flotillin-1 and alpha-tubulin. Both alpha- and beta-tubulin were detected in the rat brain raft fraction as abundant proteins, which co-immunoprecipitate with flotillin-1 and caveolin-1. Importantly, some protein-protein interactions in rafts were disrupted in 3-NY-containing proteins, e.g. caveolin-1 was dissociated from a complex with flotillin-1 and alpha-tubulin. The analysis of age dependencies did not show any significant change in protein nitration and expression of flotillin-1 and alpha-tubulin, but a decrease in the brain caveolin-1 level for old (34 months) versus young (6 months) rats. The putative mechanism of nitric oxide synthase (NOS) activity regulation by the level of caveolin expression and raft protein-protein interactions is discussed.
Dremina
Elena S
ES
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
Sharov
Victor S
VS
Schöneich
Christian
C
D000375:Aging / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D051381:Rats; D011914:Rats, Inbred BN; D011916:Rats, Inbred F344; D014404:Tubulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D014404:Tubulin; C106354:flotillins; C002744:3-nitrotyrosine; D014443:Tyrosine