Farina Biancamaria, Doti Nunzianna, Pirone Luciano, Malgieri Gaetano, Pedone Emilia M, Ruvo Menotti, Fattorusso Roberto
Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.
Biochim Biophys Acta. 2013 Aug;1834(8):1572-80. doi: 10.1016/j.bbapap.2013.04.012. Epub 2013 Apr 19.
PED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses.
PED/PEA15是一种参与多种蛋白质-蛋白质相互作用的小蛋白，它调节许多参与主要细胞功能的关键细胞效应器的功能，包括细胞凋亡、增殖和葡萄糖代谢。特别是，PED/PEA15与磷脂酶D（PLD）同工型1和2相互作用，增加蛋白激酶C-α同工型的活性，并影响胰岛素刺激的葡萄糖转运和葡萄糖刺激的胰岛素分泌。PLD1的C末端部分（第712-1074位氨基酸），称为D4，仍然能够与PED/PEA15相互作用。在本研究中，我们通过核磁共振光谱对PED/PEA15与D4α的分子相互作用进行了表征，D4α是D4的一个较小区域，包含第712-818位氨基酸，已证明其对PED/PEA15具有相同的亲和力，并在过表达PED/PEA15的细胞中诱导与D4相同的效应。化学位移扰动（CSP）研究有助于确定PED/PEA15的D4α结合位点，并识别一个可能受变构效应影响的较小区域。此外，类似酶联免疫吸附测定（ELISA）的实验表明，覆盖D4α 762-801区域的三个20聚体重叠合成肽通过与PED/PEA15结合，以低微摩尔范围的解离常数（KDs）强烈抑制PED/PEA15-D4α相互作用。最后，通过CSP和饱和转移差异（STD）分析揭示了PED/PEA15与三种肽之一相互作用的分子细节。
