Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.
It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether ... CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.
Mesh Terms:
Acetylcysteine, Adenosine Triphosphate, Animals, Blotting, Western, COS Cells, Cell Line, Cysteine Proteinase Inhibitors, DNA, Complementary, Detergents, Dose-Response Relationship, Drug, Glutathione Transferase, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Humans, Immunoglobulin G, Immunoprecipitation, Lactones, Macrolides, Nitric Oxide Synthase, Nitric Oxide Synthase Type I, Palmitic Acids, Proteasome Endopeptidase Complex, Protein Structure, Tertiary, Rabbits, Rats, Time Factors, Transfection, Ubiquitin, Ubiquitin-Protein Ligases
Acetylcysteine, Adenosine Triphosphate, Animals, Blotting, Western, COS Cells, Cell Line, Cysteine Proteinase Inhibitors, DNA, Complementary, Detergents, Dose-Response Relationship, Drug, Glutathione Transferase, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Humans, Immunoglobulin G, Immunoprecipitation, Lactones, Macrolides, Nitric Oxide Synthase, Nitric Oxide Synthase Type I, Palmitic Acids, Proteasome Endopeptidase Complex, Protein Structure, Tertiary, Rabbits, Rats, Time Factors, Transfection, Ubiquitin, Ubiquitin-Protein Ligases
J. Biol. Chem.
Date: Dec. 17, 2004
PubMed ID: 15466472
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