Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase

J Biol Chem. 2004 Dec 17;279(51):52970-7. doi: 10.1074/jbc.M406926200. Epub 2004 Oct 4.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcysteine / analogs & derivatives*
  • Acetylcysteine / metabolism
  • Adenosine Triphosphate / metabolism
  • Animals
  • Blotting, Western
  • COS Cells
  • Cell Line
  • Cysteine Proteinase Inhibitors / pharmacology
  • DNA, Complementary / metabolism
  • Detergents / pharmacology
  • Dose-Response Relationship, Drug
  • Glutathione Transferase / metabolism
  • HSP70 Heat-Shock Proteins / metabolism
  • HSP90 Heat-Shock Proteins / metabolism*
  • Humans
  • Immunoglobulin G / chemistry
  • Immunoprecipitation
  • Lactones / pharmacology
  • Macrolides
  • Nitric Oxide Synthase / metabolism*
  • Nitric Oxide Synthase Type I
  • Palmitic Acids / metabolism
  • Proteasome Inhibitors
  • Protein Structure, Tertiary
  • Rabbits
  • Rats
  • Time Factors
  • Transfection
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / physiology*

Substances

  • Cysteine Proteinase Inhibitors
  • DNA, Complementary
  • Detergents
  • HSP70 Heat-Shock Proteins
  • HSP90 Heat-Shock Proteins
  • Immunoglobulin G
  • Lactones
  • Macrolides
  • Palmitic Acids
  • Proteasome Inhibitors
  • Ubiquitin
  • lactacystin
  • Adenosine Triphosphate
  • NOS1 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type I
  • Nos1 protein, rat
  • STUB1 protein, human
  • Ubiquitin-Protein Ligases
  • Glutathione Transferase
  • monorden
  • Acetylcysteine