S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.
Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.
Martínez-Ruiz
Antonio
A
Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado, 4, E-28029 Madrid, Spain.
Villanueva
Laura
L
González de Orduña
Cecilia
C
López-Ferrer
Daniel
D
Higueras
María Angeles
MA
Tarín
Carlos
C
Rodríguez-Crespo
Ignacio
I
Vázquez
Jesús
J
Lamas
Santiago
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001710:Biotin; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010126:Ozone; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D026403:S-Nitrosothiols / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D018841:HSP90 Heat-Shock Proteins; D026403:S-Nitrosothiols; D010126:Ozone; D001710:Biotin; C021315:S-nitrosocysteine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D000251:Adenosine Triphosphatases; D003545:Cysteine