G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Science, 690022 Vladivostok, Russia.
Michael Sars Centre, University of Bergen, 5020 Bergen, Norway.
Mar Drugs. 2023 Mar 20;21(3):192. doi: 10.3390/md21030192.
Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.
嘌呤能 P2X7 受体 (P2X7) 现已被证明在许多病理条件中发挥重要作用，并代表一个重要的治疗靶点，包括神经退行性变。在这里，我们通过体外模型中的 P2X7 亚型研究了肽对神经细胞中嘌呤能信号的影响。我们发现，许多重组肽，海葵 Kunitz 型肽的类似物，能够影响高浓度 ATP 的作用，从而减少 ATP 的毒性作用。钙内流以及荧光染料 YO-PRO-1 的荧光明显被研究的肽抑制。免疫荧光实验证实，这些肽可降低神经元Neuro-2a 细胞中 P2X7 的表达水平。两种选定的活性肽，HCRG1 和 HCGS1.10，被发现可特异性地与 P2X7 的细胞外结构域相互作用，并在表面等离子体共振实验中与受体形成稳定的复合物。分子对接方法使我们能够确定最活跃的 HCRG1 肽在 P2X7 同源三聚体的细胞外结构域上的假定结合位点，并提出一种调节其功能的机制。因此，我们的工作表明 Kunitz 型肽通过影响 P2X7 受体的信号转导而具有防止神经元死亡的能力。
