Interrogating the Regulatory Importance of Protein Arginine Deiminases

描述(由申请人提供)：PTMS作为蛋白质-蛋白质相互作用和蛋白质稳定性的额外调节层，对下游信号通路产生影响，包括基因转录、细胞生长和细胞凋亡。瓜氨酸化或脱胺是 依赖钙的蛋白质精氨酸脱亚胺酶(PAD1-4和PAD6)负责将精氨酸转化为瓜氨酸。这一修改 转换胍基的正电荷，并将其替换为中性的脲基团。虽然瓜氨酸化的生理作用仍不清楚，但这种PTM已知 调节一些过程，包括网络形成和细胞分化。 Coonrod团队最近的一份报告发现，组蛋白H3R26是PAD2在多个细胞系中的体内靶点。17-雌二醇(E_2)对雌激素受体(ER)的刺激促进了该部位的瓜氨酸化。下游效应包括染色质解缩和在ER(1)控制下200多个基因的转录激活。有趣的是，与PAD4不同的是，PAD2不包含规范的核定位信号(NLS，56-PPAKKST-63)，我们假设进入细胞核的运输是通过蛋白质-蛋白质相互作用促进的。PAD2的晶体结构显示了几个潜在的对接位置，包括两个N末端免疫球蛋白样结构域和下文(2)描述的苯丙氨酸(FF)手指基序。目的1重点研究独特的Ff基序，因为初步数据表明，它通过作为一个未知调节蛋白(S)的对接位点来调节PAD2的细胞活性。为了解决这一假设，我们将采取组合和集成的方法，包括生化和蛋白质组学实验，旨在鉴定这些新的多氯联苯。 在目标2中，我们将解决第二个假设，该假设侧重于蛋白质瓜氨酸化的“读取者”存在的概念，以解释蛋白质瓜氨酸化并促进对瓜氨酸化的生理反应。总而言之，这些研究将增加我们对PAD活动调节机制的理解。因此，我们希望我们的研究将确定这些酶在细胞中被激活的条件，从而加强我们对蛋白质瓜氨酸化的生理作用的理解。我们还期望这些研究将有助于确定 应作为抑制目标的酶的精确分子结构，从而促进未来开发针对这些酶的化学探针和疗法的努力。

DESCRIPTION (provided by applicant): PTMs act as an added layer of regulation for protein-protein interactions and protein stability with consequential effects on downstream signaling pathways including gene transcription, cell growth, and apoptosis. Citrullination or deimination is one such PTM in which the calcium-dependent Protein Arginine Deiminases (PADs 1-4 and 6) are responsible for the conversion of peptidyl-arginine into peptidyl-citrulline. This modification converts the positive charge of the guanidinium group and replaces it with a neutral ureido group. Although the physiological effects of citrullination are still unclear, this PTM is known to regulate a number of processes including NET formation and cellular differentiation. A recent report by the Coonrod group identified Histone H3R26 as an in vivo target of PAD2 in multiple cell lines. Citrullination at this site is promoted by 17¿-estradiol (E2) stimulation of estrogen receptor ¿ (ER¿). Downstream effects include chromatin decondensation and transcriptional activation of more than 200 genes under the control of ER¿(1). Interestingly, unlike PAD4, PAD2 does not contain a canonical nuclear localization signal (NLS, 56-PPAKKKST-63) and we hypothesize that transport into the nucleus is facilitated by protein-protein interactions. Crystallographic structures of PAD2 show several potential docking sites, including two N-terminal immunoglobulin-like domains and the phenylalanine (FF) finger motif described below(2). Aim 1 focuses on the unique FF-motif as preliminary data suggests it regulates the cellular activity of PAD2 by acting as a docking site for an unidentified regulatory protein(s). To address this hypothesis, we will take a combinatorial and integrated approach that involves biochemical and proteomic experiments designed to identify these novel PBPs. In Aim 2, we will address a second hypothesis that focuses on the notion that 'readers' of protein citrullination exist to interpret protein citrullination and facilitate physiological resposes to citrullination. In total, these studies will increase our understanding of the mechanisms that regulate PAD activity. As a consequence, we expect that our studies will identify the conditions under which these enzymes are activated in cells thereby enhancing our understanding of the physiological roles of protein citrullination. We also expect that these studies will help identify the precise molecular structure of the enzyme that should be targeted for inhibition, thereby, facilitating future efforts to develop chemical probes and therapeutics targeting these enzymes.