Received for publication March 5, 2004.
Revised June 9, 2004.
Accepted for publication July 2, 2004.

UBIQUITINATION AND DEGRADATION OF NEURONAL NO-SYNTHASE IN VITRO: DIMER STABILIZATION PROTECTS THE ENZYME FROM PROTEOLYSIS

Abstract

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS as well as the inhibition of hsp90-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient apo-nNOS is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of apo-nNOS and enhance its degradation in vivo. In the current study we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with N^G-nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it appears that the dimeric structure is a major determinant of nNOS stability and proteolysis.

Key words: Nitric oxide synthases, Regulation - post-transcriptional, Reactive intermediates

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