Ubiquitination and Degradation of Neuronal Nitric-Oxide 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 and the inhibition of heat shock protein 90 (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 nNOS (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 aponNOS 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 seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.

Tetrahydrobiopterin (BH4), a cofactor for nNOS, restores gastric emptying and nNOS expression in female diabetic rats

“…Supplementation of BH4 stabilizes the functionally active, dimeric form of nNOS in vitro. BH4 also inhibits monomerization of nNOS, as well as inactivation of the enzyme (11,20). Our in vivo data, for the first time, have shown that dietary BH4 supplementation restores both nNOS dimerization and enzyme activity in diabetic rat stomach.…”

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

“…In insulin-resistant skeletal muscle, we found that nNOS was actively degraded by the proteasome [37], as demonstrated by the presence of high amounts of ubiquitinated nNOS and by the effect of the inhibitor MG 132, which was able to restore nNOS expression and catalytic activity to a normal level. We also detected lower amounts of SDS-resistant dimers in fa/fa vs fa/+ skeletal muscle (data not shown), which might favour increased susceptibility to proteasomal degradation; indeed, dimerisation is known to protect nNOS from proteolysis [38]. The specific signals targeting nNOS to the proteasome have not been clearly identified.…”

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity

“…nNOS overexpression could be due to an enhanced activity of transcription factors, as nuclear factor kappa-B (NFκB) and cAMP response element (CRE) are reportedly involved in nNOS induction [24][25][26]. In addition, the dimeric conformation of nNOS has been shown to protect the protein from proteolysis by the ubiquitinproteasome pathway [27]. However, despite a sevenfold increase in nNOS protein level, we found only a 2.7-fold increase in phospho-Ser847 in fa/fa rats, which plays probably only a minor role in the relative reduction of nNOS activity.…”