作为一系列信号事件中的关键调解人,磷酸肌醇(PIPs)在精确的时刻将蛋白质招募到特定的细胞位置。蛋白质活性的这种复杂的时空调节通常需要相应的PIP的局部富集。我们研究了磷脂酰肌醇-4-磷酸酯(PI(4)P)的程度和热稳定性,磷脂酰肌醇-4,5-二磷酸(PI(4,5)P2和磷脂酰肌醇-3,4,5-三磷酸(PI(3,4,5)P3)与钙和镁离子簇。在存在Mg2+离子的情况下,我们观察到所有检查的PIP的可忽略或最小的聚集。而PI(4)P显示在Ca2+的存在下没有聚集,PI(4,5)P2与至少在80°C下表现出稳定性的Ca2+强簇形成。PI(3,4,5)P3与Ca2+相互作用的簇形成程度小于PI(4,5)P2的簇形成程度,但我们仍然观察到一些高达80°C的簇。鉴于胆固醇已被证明可以增强PIP聚类,我们研究了二价阳离子和胆固醇是否协同促进PIP聚集。我们发现Mg2+或Ca2+与PI(4)P的相互作用仍然非常弱,即使是在胆固醇的存在下。相比之下,我们观察到胆固醇和Ca2+与PI(4,5)P2的协同相互作用。此外,在胆固醇的存在下,Mg2+与PI(4,5)P2的相互作用仍然较弱。在我们研究的实验条件下,PI(3,4,5)P3未显示出与胆固醇的强聚类,并且与Ca2和Mg2的相互作用不受胆固醇的存在的影响。
As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.