PEROXYNITRITE INCREASES THE DEGRADATION OF ACONITASE AND OTHER CELLULAR PROTEINS BY PROTEASOME

We report that exposure of aconitase to moderate concentrations of per oxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide-and nitric ox ide-liberating substance), or hydrogen peroxide, inhibits the enzyme a nd enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, fro m these same agents, causes further inhibition of the enzymatic activi ty of aconitase but actually decreases its proteolytic breakdown by pr oteasome. It should be noted that the superoxide and nitric oxide libe rated by SIN-1 decomposition react to form a steady flux of peroxynitr ite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or l ess) and has no effect on the proteolytic susceptibility of the enzyme . Proteasome also seems to be the main protease in cell lysates that c an degrade aconitase after it has been oxidatively modified by exposur e to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates is olated from K562 cells treated for several days with an antisense olig odeoxynucleotide to the initiation codon region of the C2 subunit of p roteasome (a treatment which diminishes proteasome activity by 50-60%) , the enhanced degradation of moderately damaged aconitase was essenti ally abolished. Other model proteins as well as complex mixtures of pr oteins, such as cell lysates, also exhibit enhanced proteolytic suscep tibility after moderate SIN-1 treatment. Therefore we conclude that pe roxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by pro teasome.