C331A mutant of neuronal nitric-oxide synthase is labilized for Hsp70/CHIP (C terminus of HSC70-interacting protein)-dependent ubiquitination.
It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N(G)-nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.
Clapp
Kelly M
KM
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Peng
Hwei-Ming
HM
Morishima
Yoshihiro
Y
Lau
Miranda
M
Walker
Vyvyca J
VJ
Pratt
William B
WB
Osawa
Yoichi
Y
D020134:Catalytic Domain; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry; D009154:Mutation; D052248:Nitric Oxide Synthase Type I / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D015262:Xenobiotics / Q000737:chemistry
D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008024:Ligands; D018832:Molecular Chaperones; D025801:Ubiquitin; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D052248:Nitric Oxide Synthase Type I; D044767:Ubiquitin-Protein Ligases