Labilization and proteasomal degradation of neuronal nitric oxide synthase.

Abstract

The nitric oxide synthases (NOS) are cytochrome P450-like heme-containing enzymes that catalyze the production of nitric oxide and L-citrulline from L-arginine. Our laboratory has shown that certain drugs, such as guanabenz, inactivate neuronal NOS (nNOS) and lead to the selective ubiquitination and proteasomal degradation of the dysfunctional enzyme. The exact mechanism of how this occurs is unknown. In this thesis, I determined the mechanism of inactivation of nNOS by guanabenz with the use of a purified enzyme and developed an <italic>in vitro</italic> degradation model to better understand how dysfunctional nNOS is selectively degraded. I found that guanabenz inactivates nNOS in a time- and concentration-dependent manner. The loss of activity is concomitant with oxidative destruction of tetrahydrobiopterin (BH₄) and destabilization of the dimeric form of nNOS. Guanabenz also causes heme destruction <italic> in vitro</italic> if antioxidant proteins are absent. However, in rats the effect of guanabenz is completely blocked by coadministration of BH₄, suggesting that BH₄ loss and dimer destabilization are the major mechanisms for activity and protein loss <italic>in vivo</italic>. Consistent with the guanabenz result <italic>in vivo</italic>, destabilization of the dimeric form of nNOS is sufficient for degradation in an <italic>in vitro</italic> system containing partially purified reticulocyte proteins. This degradation is dependent upon ubiquitin, ATP, and the proteasome. Degradation occurs whether or not heme is bound to nNOS. Finally, the competitive and reversible inhibitors of nNOS, 7-nitroindazole and N<super>G</super>-nitro-L-arginine, stabilize the nNOS dimer and decrease degradation of the protein. Taken together, these data suggest that dimer destabilization is a major determinant for the degradation of nNOS <italic>in vivo</italic>. Thus, drugs that favor nNOS dimer destabilization cause the rapid proteolysis nNOS <italic>in vivo</italic>. This work helps to explain how xenobiotics affect protein levels within the cell and lead to potential drug toxicities and side effects.