Bender AT, Demady DR, Osawa Y. 2000. Ubiquitination of neuronal nitric oxide synthase in vitro and in vivo. J Biol Chem 275(23):17407–17411.
It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of 125I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme.
Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion ofL-arginine to citrulline and nitric oxide by a process that requires NADPH and molecular oxygen (1-4). The enzymes also require bound FMN, FAD, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), and Ca2+/calmodulin for activity. Three main isoforms of NOS have been identified: isoform I or neuronal NOS (nNOS), which is constitutively expressed in a variety of neuronal cells as well as other cells; isoform II or inducible NOS, which is usually not constitutively expressed but can be induced by bacterial lipopolysaccharide and/or cytokines in macrophages and other cells; and isoform III or endothelial NOS, which is expressed in endothelial cells (5, 6).
Both nNOS and endothelial NOS are hsp90-associated proteins and inhibition of hsp90 by geldanamycin causes the loss of NOS protein in cells (7, 8). Based on studies with other hsp90-associated proteins, this loss of NOS is likely due to enhanced proteasomal degradation, presumably as a mechanism for the removal of misfolded proteins (9-11). It has been shown for some proteins, including p53 (11) and tyrosine kinase p185c-erbB-2 (12), that geldanamycin treatment leads to the accumulation of ubiquitinated forms of the target protein. More recently, our laboratory has shown that suicide inactivation of nNOS enhances the proteolytic removal of the enzyme that is mediated, in part, by the proteasome (13). Based on these studies, we asked if nNOS could be regulated by ubiquitination prior to proteolytic removal by the proteasome in HEK 293 cells transfected with nNOS. This cellular model was chosen since it is the same model used in studies on the suicide inactivation and hsp90 regulation of nNOS (7, 13).
In the current study, we have found that inhibition of the proteasome leads to the accumulation of higher molecular mass forms of nNOS, which are in part due to conjugation with ubiquitin (Ub). The major nNOS-Ub conjugate did not greatly change the relative mobility of the protein on SDS-PAGE gels, suggesting that the conjugate contains a few or perhaps one Ub. This limited ubiquitination of nNOS could be reproduced with an in vitro system containing purified nNOS, reticulocyte extracts, ATP, and Ub. Studies with 125I-Ub indicate that the heme-deficient monomeric form of nNOS is preferentially ubiquitinated over the dimeric active form of nNOS. Thus, these studies establish for the first time that ubiquitination of nNOS occurs and likely plays a regulatory role in the removal of misfolded or non-functional protein.