title: Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase.
abstract: 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.
authors.affiliation: Institute of Bioorganic Chemistry, Academy of Sciences Siberian Division, Novosibirsk, Russia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Laktionov
authors.forename: Elena
authors.initials: E
authors.lastname: Rykova
authors.forename: Mattia
authors.initials: M
authors.lastname: Toni
authors.forename: Enzo
authors.initials: E
authors.lastname: Spisni
authors.forename: Cristiana
authors.initials: C
authors.lastname: Griffoni
authors.forename: Anton
authors.initials: A
authors.lastname: Bryksin
authors.forename: Natalia
authors.initials: N
authors.lastname: Volodko
authors.forename: Valentin
authors.initials: V
authors.lastname: Vlassov
authors.forename: Vittorio
authors.initials: V
authors.lastname: Tomasi
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2
chemical_list: D016376:Oligonucleotides, Antisense; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010741:Phospholipases A; D054467:Phospholipases A2