Ubiquitination of neuronal nitric-oxide synthase in vitro and in vivo.
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 (125)I-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.
Bender
A T
AT
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
Demady
D R
DR
Osawa
Y
Y
D000818:Animals; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007668:Kidney; D009097:Multienzyme Complexes / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000302:isolation & purification / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014452:Ubiquitins / Q000302:isolation & purification / Q000378:metabolism*
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014452:Ubiquitins; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex