Pradhan Apoorva J, Chitkara Shweta, Ramirez Ricardo X, Monje-Galvan Viviana, Sancak Yasemin, Ekin Atilla-Gokcumen G
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA.
Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA.
bioRxiv. 2023 Nov 2:2023.08.19.553906. doi: 10.1101/2023.08.19.553906.
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 the 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 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, C184, C269 and C286 are the possible acylation sites. Using all atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane level. Through systematic investigations of the -palmitoyltransferases that might acylate MLKL 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 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转位至质膜并诱导膜通透性增加，这会导致与坏死性凋亡相关的炎症。MLKL与膜的结合最初是由该蛋白与膜磷脂之间的静电相互作用引发的。我们之前表明，MLKL及其磷酸化形式（pMLKL）在坏死性凋亡过程中会发生酰化。在此，我们对MLKL的酰化位点进行了表征，并确定了多个可发生酰化的半胱氨酸，且存在有趣的混杂情况。我们的结果表明，MLKL和pMLKL在单个半胱氨酸C184处发生酰化，C269和C286是可能的酰化位点。通过全原子分子动力学模拟，我们确定了MLKL酰化在蛋白质和膜水平上所导致的差异。通过对可能在坏死性凋亡中使MLKL发生酰化的棕榈酰转移酶进行系统研究，我们发现zDHHC21的活性对pMLKL酰化的影响最强，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的酰化会使该蛋白在膜界面处不稳定并导致其降解，从而减轻坏死性凋亡活性。从更广泛的层面来看，我们的研究结果揭示了酰化对MLKL在坏死性凋亡中的功能以及由其酰化介导的MLKL与膜相互作用的影响。
