Identification of Actin as a 15-Deoxy-Δ^12,14-prostaglandin J₂Target in Neuroblastoma Cells:  Mass Spectrometric, Computational, and Functional Approaches To Investigate the Effect on Cytoskeletal Derangement

Abstract

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.

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

“…In the present study, we demonstrated that IL-3 stimulates expression of 14-3-3␥, which induces resistance to apoptotic death and promotes proliferation of IL-3-dependent Ba/F3 cells. The data also indicated that overexpression of 14-3-3␥ may promote neoplastic transformation, and that down-regulation of 14-3-3␥ in transformed hematopoietic cell lines can lead to growth control and death, potentially providing a new target for cancer therapy (6,36,41,42).…”

Cyclopentenone Prostaglandins with Dienone Structure Promote Cross-Linking of the Chemoresistance-Inducing Enzyme Glutathione Transferase P1-1

“…cyPGs possess an ␣,␤-unsaturated carbonyl group in the cyclopentane ring, a structure that favors the formation of Michael adducts with thiol groups in proteins or in GSH. Among the proteins known to be targets for modification by addition of cyPG are transcription factors, such as activator protein-1 and nuclear factor-B (Cernuda-Morollón et al, 2001;Pérez-Sala et al, 2003); proteins involved in the regulation of cellular redox status, such as thioredoxin and thioredoxin reductase (Shibata et al, 2003;Cassidy et al, 2006); and cytoskeletal proteins (Stamatakis et al, 2006;Aldini et al, 2007), among others.…”

Sepsis-induced inhibition of neutrophil chemotaxis is mediated by activation of peroxisome proliferator-activated receptor-γ

“…35 The reasons for the disparate inhibitory effects on actin polymerization between troglitazone and 15d-PGJ 2 in this study remain unclear, but possibly reflect the complexity of interactions between 15d-PGJ 2 and actin. One study showed that 15d-PGJ 2 interacts directly with the actin molecule in neuroblastoma cells to depolymerize F-actin and block polymerization, but only modest effects were seen at a concentration of 20 M. 36 We tested only concentrations of 5 and 10 M because these concentrations had proved adequate to inhibit chemotaxis.The rapid effects observed in this study occur in a shorter time span than is typical for effects mediated by nuclear transcription. This might be viewed as arguing for a PPAR-␥-independent mechanism despite similar effects shown by 2 structurally dissimilar ligands and by a constitutively active PPAR-␥ construct.…”