Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase

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• 作者：Laktionov ; Pavel ; Rykova ; Elena ; Toni ; Mattia ; Spisni ; Enzo ; Griffoni ; Cristiana ; Bryksin ; Anton ; et. al.
• 关键词：cPLA2 ; Antisense oligonucleotide ; Glyceraldehyde-3-phosphate dehydrogenase
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids
• 出版年：2004
• 出版时间：March 22, 2004
• 年：2004
• 卷：1636
• 期：2-3
• 页码：129-135
• 全文大小：405 K

文摘

We have previously shown that an antisense, effective in the knock down of cytosolic phospholipase A2 (cPLA2), localizes mainly in the nucleus of human endothelial cells and monocytes and that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in its nuclear localization. In this study, we clarify how GAPDH participates in the nuclear localization of this antisense oligodeoxynucleotide (ODN) directed against cPLA2 mRNA. A central TAAAT motif providing specificity and high affinity binding was assumed to interact with the enzyme Rossmann fold region on the basis of competition to this site by NAD⁺. To asses whether the TAAAT motif interacts directly with the enzyme Rossmann fold region, we evaluated the binding to GAPDH of different oligonucleotides and the effect of competitors such as NAD⁺, NADH, mononucleotides, DNA, polyribonucleic acids and polyanions. We found that the dissociation constant for TAAAT containing oligonucleotides was three - to fivefold higher with respect to oligo not containing this motif. By covalently linking ³²P-labeled cPLA2p(N)₁₆ to GAPDH and after executing hydrolysis with hydroxylamine, the labeling was exclusively found in the C-terminal domain (aa 286–334). These results indicate that the antisense oligonucleotide interacts with a site not having a defined function but which can be negatively allosterically regulated when NAD⁺ or polynucleotides are bound to Rossmann fold.