Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15.
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 HxKx(4)Dx(6)GSxN 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-His(6)-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His(6)-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.
Viparelli
Francesca
F
Istituto di Biostrutture e Bioimmagini (IBB), CNR, via Mezzocannone 16, 80134 Napoli, Italy.
Doti
Nunzianna
N
Sandomenico
Annamaria
A
Marasco
Daniela
D
Dathan
Nina A
NA
Miele
Claudia
C
Beguinot
Francesco
F
Monti
Simona M
SM
Ruvo
Menotti
M
D051017:Apoptosis Regulatory Proteins; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D010739:Phospholipase D / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary / Q000235:genetics; D020349:Surface Plasmon Resonance
D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; C092039:PEA15 protein, human; D010750:Phosphoproteins; D010739:Phospholipase D; C106801:phospholipase D1