title: 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 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 NG-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.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, 1301 Medical Science Research Building III, Ann Arbor 48109-0632, USA.
authors.forename: Anwar Y
authors.initials: AY
authors.lastname: Dunbar
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kamada
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Jenkins
authors.forename: Ezra R
authors.initials: ER
authors.lastname: Lowe
authors.forename: Scott S
authors.initials: SS
authors.lastname: Billecke
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Osawa
mesh_terms: D000818:Animals; D001673:Biodegradation, Environmental; D019281:Dimerization; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011817:Rabbits; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D006418:Heme; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex