Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement.
A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.
Aldini
Giancarlo
G
Istituto di Chimica Farmaceutica e Tossicologica Pietro Pratesi, Faculty of Pharmacy, University of Milan, I-20131 Milan, Italy. giancarlo.aldini@unimi.it
Carini
Marina
M
Vistoli
Giulio
G
Shibata
Takahiro
T
Kusano
Yuri
Y
Gamberoni
Luca
L
Dalle-Donne
Isabella
I
Milzani
Aldo
A
Uchida
Koji
K
D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D019295:Computational Biology / Q000379:methods*; D003599:Cytoskeleton; D016503:Drug Delivery Systems; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D009447:Neuroblastoma / Q000473:pathology; D015230:Prostaglandin D2 / Q000008:administration & dosage / Q000031:analogs & derivatives*; D014407:Tumor Cells, Cultured
D000199:Actins; C037112:9-deoxy-delta-9-prostaglandin D2; D015230:Prostaglandin D2