Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States.
Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States.
ACS Chem Biol. 2024 Feb 16;19(2):407-418. doi: 10.1021/acschembio.3c00603. Epub 2024 Feb 1.
Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization, which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are -acylated during necroptosis. Here, we characterize the acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, with C184, C269, and C286 as possible acylation sites. Using all-atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane levels. Through investigations of the -palmitoyltransferases that might acylate pMLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating the necroptotic activity. At a broader level, our findings shed light on the effect of -acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.
混合谱系激酶结构域样蛋白（MLKL）是坏死性凋亡的关键信号蛋白。在磷酸化激活后，MLKL 易位到质膜并诱导膜通透性增加，这有助于坏死性凋亡相关的炎症。MLKL 与质膜的最初结合是通过蛋白质与膜磷脂之间的静电相互作用起始的。我们之前曾表明，在坏死性凋亡过程中，MLKL 及其磷酸化形式（pMLKL）被酰化。在这里，我们对 MLKL 的酰化位点进行了表征，并确定了多个半胱氨酸，这些半胱氨酸可能具有有趣的酰化多效性。我们的结果表明，MLKL 和 pMLKL 在单个半胱氨酸上发生酰化，C184、C269 和 C286 可能是酰化位点。通过全原子分子动力学模拟，我们确定了酰化 MLKL 在蛋白质和膜水平上引起的差异。通过研究可能在坏死性凋亡中酰化 pMLKL 的 -棕榈酰转移酶，我们表明 zDHHC21 活性对 pMLKL 的酰化作用最强，其失活会显著降低细胞中 pMLKL 的水平并改善膜的完整性。这些结果表明，阻断 pMLKL 的酰化会使质膜界面处的蛋白质不稳定并导致其降解，从而改善坏死性凋亡活性。更广泛地说，我们的发现揭示了 -酰化对 MLKL 在坏死性凋亡中的功能以及其酰化介导的 MLKL-膜相互作用的影响。
