Molecular mechanisms of lithium action on kinases

摘要/摘要 锂是数百万患有躁郁症的人的一线治疗方法，并有望 抑制痴呆症的发展。实验表明，LI改变生理的主要方式是 是通过减少数量惊人的依赖于镁的磷酰化转移的活性来实现的 酶，包括磷酸单酯酶和蛋白激酶。而(不依赖于锂的)催化剂 这些酶的机制已经被很好地理解了，很大程度上是关于它们 锂的易感性仍不清楚。毫不奇怪，设计酶变体仍然是一个重大挑战。 是抗LI的，并用它们来解开与个体LI易感性相关的信号通路 酵素。在这里，我们将重点放在李S对肌动蛋白的作用上，并解决以下问题，以缓解 以上提出的问题。对71个人的激酶进行的实验表明，锂的敏感性范围很广-许多是 没有受到影响，其他人受到不同程度的影响。但对于这些变化，没有任何解释。 我们通过使用最先进的分子力学来解决我们对锂作用力的理解上的这个空白 (Mm)、量子力学(QM)和QM/MM模拟，以及由 生物信息学和自然选择。在实验的支持下，我们探索了最重要的假设 通过直接与其催化部位相互作用来影响激酶的活性。在目标1中，模拟将检查LI如何 结合激酶，以及Li结合是如何降低激酶活性的。此外，模拟将提供对 调节催化部位活性的潜在变构效应。我们的生化、细胞和活体实验 在AIM2中被设计用来(I)系统地检测LI敏感和LI- 抗性激酶，目标是制造一种对锂敏感的酶，GSK-3，对锂产生抗性；以及(Ii)发现关键 使某些酶对锂敏感的残基。实验还将验证模拟的结果，并在 同时，模拟将提供分子洞察力来解释突变实验的结果。 对模拟和实验结果的综合分析将产生一种抗锂的GSK-3，它是 意义重大，因为它将首次使我们能够解开GSK-3驱动的LI的生理效应 与其他对锂敏感的酶不同。这项研究也将提供物理基础来解释观察到的 这些生物物理发现将成为未来研究的基础 努力使其他对锂敏感的激酶对锂产生抗性，并绘制其特定的表型图。我们期待着 这些努力将改善对LI治疗和剂量的患者反应的理解和预测， 这仍然是一项艰巨的任务。这将既加快治疗速度，又避免暴露于副作用。最后，这一点 这项研究将探索模拟酶反应的新进展，并产生经过验证的极化力 用于描述Li/Mg2与蛋白质相互作用的字段。这将使未来对锂作用的可靠研究成为可能。 在这个项目中没有考虑到的蛋白质，并扩大了对全系列镁结合蛋白质的探索。

Summary/Abstract Lithium is a first-line therapy for millions of people suffering from bipolar disorder, and is promising for inhibiting development of dementia. Experiments show that a primary mode by which Li+ alters physiological processes is by reducing activities of a surprisingly limited number of Mg2+-dependent phosphoryl-transferring enzymes, including phosphomonoesterases and protein kinases. While the (Li-independent) catalytic mechanisms of these enzymes are quite well-understood, much about the mechanistic details underlying their Li-susceptibility remain unknown. Not surprisingly, it remains a major challenge to design enzyme variants that are Li-resistant, and use them to disentangle signaling pathways associated with Li-susceptibilities of individual enzymes. Here we focus on Li+'s action on kinases, and address the following problem central to alleviating the issues raised above. Experiments on 71 human kinases show a wide range of Li-susceptibility — many are unaffected and others are affected to varying degrees. But there is no explanation for these variations. We address this gap in our understanding of Li-action by using state-of-the-art molecular mechanics (MM), quantum mechanics (QM) and QM/MM simulations, as well as mutagenesis experiments guided by bioinformatics and natural selection. Supported by experiments, we explore the overarching hypothesis that Li+ affects kinase activity by interacting directly with their catalytic sites. In Aim 1, simulations will examine how Li+ binds kinases, and how Li+ binding reduces kinase activity. Additionally, simulations will provide insights into potential allosteric effects that regulate catalytic site activity. Our biochemical, cellular and in vivo experiments in Aim2 are designed to (i) systematically examine effects of sequence differences between Li-sensitive and Li- resistant kinases, with the goal of making a Li-sensitive enzyme, GSK-3, resistant to Li+; and (ii) discover key residues that make certain kinases Li-sensitive. Experiments will also validate findings from simulations, and at the same time, simulations will provide molecular insights to interpret results from mutational experiments. Combined analysis of results from simulations and experiments will yield a Li-resistant GSK-3, which is significant because it will, for the first time, enable us to disentangle GSK-3-driven physiological effects of Li+ from those of other Li-sensitive enzymes. This study will also provide a physical basis to explain observed variations of Li-sensitivity across kinases, and these biophysical findings will serve as foundations for future efforts to make other Li-sensitive kinases resistant to Li+, and map their specific phenotypes. We expect that such efforts will improve understanding and predictions of patient responses to Li-treatments and dosages, which remains a difficult task. This will both expedite therapy and avoid exposure to side effects. Finally, this study will explore new advancements in modeling enzyme reactions and yield a validated polarizable force field for describing Li+/Mg2+ interactions with proteins. This will enable future reliable studies of Li-action on proteins not considered in this project and broaden exploration of the full range of Mg-binding proteins.