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.
| Original language | English |
|---|---|
| Pages (from-to) | 129-135 |
| Number of pages | 7 |
| Journal | Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids |
| Volume | 1636 |
| Issue number | 2-3 |
| DOIs | |
| Publication status | Published - Mar 22 2004 |
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Keywords
- Antisense oligonucleotide
- cPLA2
- Glyceraldehyde-3-phosphate dehydrogenase
ASJC Scopus subject areas
- Cell Biology
- Molecular Biology
- Biophysics
Cite this
Knock down of cytosolic phospholipase A2 : An antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3- phosphate dehydrogenase. / Laktionov, Pavel; Rykova, Elena; Toni, Mattia; Spisni, Enzo; Griffoni, Cristiana; Bryksin, Anton; Volodko, Natalia; Vlassov, Valentin; Tomasi, Vittorio.
In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1636, No. 2-3, 22.03.2004, p. 129-135.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Knock down of cytosolic phospholipase A2
T2 - An antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3- phosphate dehydrogenase
AU - Laktionov, Pavel
AU - Rykova, Elena
AU - Toni, Mattia
AU - Spisni, Enzo
AU - Griffoni, Cristiana
AU - Bryksin, Anton
AU - Volodko, Natalia
AU - Vlassov, Valentin
AU - Tomasi, Vittorio
PY - 2004/3/22
Y1 - 2004/3/22
N2 - 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.
AB - 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.
KW - Antisense oligonucleotide
KW - cPLA2
KW - Glyceraldehyde-3-phosphate dehydrogenase
UR - http://www.scopus.com/inward/record.url?scp=2942661924&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2942661924&partnerID=8YFLogxK
U2 - 10.1016/j.bbalip.2003.10.012
DO - 10.1016/j.bbalip.2003.10.012
M3 - Article
VL - 1636
SP - 129
EP - 135
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
SN - 1388-1981
IS - 2-3
ER -