MKP5 allostery in MAPK regulation and signaling in the heart

摘要 丝裂原活化蛋白激酶（MAPK）磷酸酶（MKPs）的催化活性和特异性是 通过与MAPKs互动的能力进行管理。MKPs决定了MAPK的保真度和动力学 通过以空间上不同的方式调节MAPK活性的幅度和持续时间来调节信号传导。 然而，这些调节机制本身不足以解释MKP/MAPK的复杂性。 介导的信号传导。这通过观察到MKP的失活递送独特的，并且在某些情况下， 在某些情况下，不明显的生理和病理生理结果导致人们认识到， MKP代表用于治疗某些疾病的有价值的治疗靶标。我们发现MKP 5， 直接使p38 MAPK和JNK去磷酸化，参与心脏纤维化的发展， 心力衰竭前的严重后遗症缺乏MKP 5的小鼠可以免受 压力超负荷模型中的心脏纤维化和心力衰竭。高通量小分子筛选 发现了MKP 5的第一种抑制剂。与以下化合物复合的抑制剂的共晶结构的解析 MKP 5显示抑制剂结合到MKP 5上的变构位点。我们假设MKP 5变构蛋白 位点代表了MKP 5调节的另一种模式，其控制信号传导特异性，并且这种变构 位点代表了MKP 5用于小分子靶向的“阿喀琉斯之踵”。这项工作的广泛目标是双重的。 首先，我们将定义变构位点如何调节MKP 5催化和MAPK的分子基础 发信号。第二，在疾病背景下，变构位点的破坏是否会产生结果， 概念证明，如果成功“下药”，这个网站代表了一流的发展平台 MKP抑制剂。我们将通过三个具体目标来落实这两个大目标的具体内容。目标一，威尔 采用结构生物学和生物物理学方法来阐明结构决定因素和结合 由MKP 5变构位点赋予的机制，以确定该位点如何调节MKP 5催化， MAPK结合。将获得MKP 5和p38 MAPK之间的共复合物，以确定p38 MAPK如何与MKP 5结合。 通过变构位点调节MKP 5。在目的2中，MKP 5变构位点中的突变体对 MAPK信号传导将通过使用产生含有MKP 5变构位点突变体的敲入细胞来确定。 CRISPR-Cas方法。MKP 5变构位点对MAPK介导的信号传导的“微调”特异性 将使用非偏倚磷酸蛋白质组学和转录组学方法来确定。在目标3中， 靶向心脏纤维化和心力衰竭中的MKP 5变构位点将通过生成 一种新的MKP 5变构位点突变敲入小鼠。这些研究将确定一种新的运作模式， MKP 5，其将在原子水平上提供关于变构位点的作用的信息。MKP 5如何 变构位点影响生理信号传导和变构位点作为“可用药”靶标的有效性， 将定义疾病疗法。

ABSTRACT The catalytic activity and specificity of the mitogen-activated protein kinase (MAPK) phosphatases (MKPs) is governed through their ability to interact with the MAPKs. The MKPs set the fidelity and kinetics of MAPK signaling by regulating, in a spatially distinct manner, both the magnitude and duration of MAPK activity. However, these regulatory mechanisms alone are insufficient to explain the complexity of MKP/MAPK- mediated signaling. This is exemplified by the observation that inactivation of MKPs deliver unique, and in some cases, non-obvious physiological and pathophysiological outcomes that has led to the realization that MKPs represent valuable therapeutic targets for the treatment of certain diseases. We have found that MKP5, which directly dephosphorylates p38 MAPK and JNK, is involved in the development of cardiac fibrosis, a serious sequalae that precedes heart failure. Mice lacking MKP5 are protected from the development of cardiac fibrosis and heart failure in a model of pressure overload. A high throughput small molecule screen identified a first-in-class inhibitor of MKP5. Resolution of the co-crystal structure of the inhibitor in complex with MKP5 revealed that the inhibitor bound to an allosteric site on MKP5. We hypothesize that the MKP5 allosteric site represents an additional mode of MKP5 regulation that governs signaling specificity and that this allosteric site represents the “Achilles heel” of MKP5 for small molecule targeting. The broad goal of this work is two-fold. First, we will define the molecular basis for how the allosteric site regulates MKP5 catalysis and MAPK signaling. Second, whether disruption of the allosteric site produces outcomes in a disease context that provide proof-of-concept that if successfully “drugged” this site represents a platform for development of first-in-class MKP inhibitors. We will execute the specifics of these two broad goals through three specific aims. Aim 1, will employ structural biology and biophysical approaches to elucidate the structural determinants and binding mechanisms conferred by the MKP5 allosteric site to determine how this site regulates MKP5 catalysis and MAPK binding. A co-complex between MKP5 and p38 MAPK will be obtained to define how p38 MAPK modulates MKP5 through the allosteric site. In Aim 2, the effects of mutants in the MKP5 allosteric site on MAPK signaling will be determined by generating knockin cells harboring MKP5 allosteric site mutants using CRISPR-Cas approaches. The specificity of the MKP5 allosteric site to “fine-tune” MAPK-mediated signaling will be determined using non-biased phosphoproteomic and transcriptomic approaches. In Aim 3, the effects of targeting the MKP5 allosteric site in cardiac fibrosis and heart failure will be determined through the generation of a novel MKP5 allosteric site mutant knock-in mouse. These studies will define a new mode of operation for MKP5 that will provide information on the actions of the allosteric site at the atomic level. How the MKP5 allosteric site impacts physiological signaling and the validity of the allosteric site as a “druggable” target in disease therapeutics will be defined.