Mechanisms of MLKL in necroptosis: from intramolecular rearrangements to isoform regulation

坏死性凋亡是一种新发现的调节性坏死形式，是炎症性的，在许多与慢性炎症和感染相关的疾病中起作用。它导致质膜透化后细胞内容物的释放，依赖于假激酶混合谱系激酶结构域样（MLKL）蛋白，这是迄今为止已知的坏死性凋亡的最末端效应子。此外，最近已经描述了MLKL在细胞内囊泡运输中的非致命作用，其似乎抵消了坏死性细胞死亡。然而，这些MLKL功能背后的分子机制仍然模糊，是一个有争议的问题。在初步工作中，我们发现通过转录组分析鉴定的小鼠和人MLKL的不同亚型表现出不同的死亡诱导效力。虽然缺乏抑制性假激酶结构域使MLKL具有内在活性，但在C-末端螺旋中仅插入8个氨基酸就消除了其坏死性凋亡活性。该项目的主要目标是阐明MLKL如何执行坏死性凋亡的分子机制。 为此，我们将揭示调节MLKL活性的结构元件，并确定不同MLKL亚型对MLKL致命和非致命功能的贡献。基于C-末端螺旋序列的微小差异对MLKL活性的影响，我们计划研究MLKL结构如何调节功能。然后，我们将使用这些信息来发现调节MLKL活性的新的小分子。最后，我们将研究不同MLKL亚型对坏死性凋亡和其他非致命功能的调节作用。这个多学科的项目结合了细胞生物学和先进的显微镜与分子动力学模拟，以揭示新的分子步骤参与协调坏死性凋亡。新的小分子和蛋白质参与坏死性凋亡调控的鉴定将为设计调节MLKL活性以促进人类健康的新药铺平道路。

Necroptosis is a newly discovered form of regulated necrosis that is inflammatory and plays a role in a number of diseases associated with chronic inflammation and infection. It results in release of the cellular contents after plasma membrane permeabilization dependent on the pseudokinase mixed lineage kinase domain-like (MLKL) protein, which is the most terminal effector of necroptosis known to date. Additionally, non-deadly roles of MLKL in intracellular vesicle trafficking that seem to counterbalance necroptotic cell death have been recently described. However, the molecular mechanism behind these MLKL functions remains obscure and is a matter of debate. In preliminary work, we have discovered that the different isoforms of mouse and human MLKL identified by transcriptome analysis exhibit distinct death-inducing potency. While lack of the inhibitory pseudokinase domain renders MLKL intrinsically active, insertion of just eight amino acids in the C-terminal helix abolishes its necroptotic activity. The main goal of this project is to shed new light on the molecular mechanism how MLKL executes necroptosis. To this aim, we will uncover the structural elements that regulate MLKL activity and determine the contribution of the different MLKL isoforms to MLKL deadly and non-deadly functions. Based on the effect of small differences in the sequence of the C-terminal helix on MLKL activity, we plan to investigate how MLKL structure regulates function. We will then use this information to discover new small molecules that regulate MLKL activity. Finally, we will examine the role of the different MLKL isoforms on the regulation of necroptosis and of additional non-deadly functions. This multidisciplinary project combines cell biology and advanced microscopy with molecular dynamic simulations to disclose new molecular steps involved in the coordination of necroptosis. The identification of new small molecules and proteins implicated in necroptosis regulation will pave the way for the design of new drugs that modulate MLKL activity for human health.