Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15 |
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Authors: | Viparelli Francesca Doti Nunzianna Sandomenico Annamaria Marasco Daniela Dathan Nina A Miele Claudia Beguinot Francesco Monti Simona M Ruvo Menotti |
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Affiliation: | aIstituto di Biostrutture e Bioimmagini (IBB), CNR, via Mezzocannone, 16, 80134, Napoli, Italy;bDipartimento di Scienze Biologiche, Università di Napoli Federico II, via Mezzocannone, 16, 80134 Napoli, Italy;cDipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via S.M. di Costantinopoli 16, 80138 Napoli, Italy;dIstituto di Endocrinologia ed Oncologia Sperimentale (IEOS), CNR, Via Pansini, 5, 80131 Napoli, Italy |
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Abstract: | PLD’s (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx4Dx6GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712–1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His6-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His6-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions. |
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Keywords: | Phospholipase Phosphoprotein enriched in diabetes (PED) SPR Protein– protein interaction |
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