Interrogating the Regulatory Importance of Protein Arginine Deiminases

描述（由申请人提供）：PTM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，对下游信号传导途径（包括基因转录、细胞生长和细胞凋亡）产生相应影响。瓜氨酸或脱亚氨基是 一种这样的PTM，其中钙依赖性蛋白质精氨酸脱亚胺酶（PAD 1-4和6）负责肽基-精氨酸转化为肽基-瓜氨酸。此修改 转化胍鎓基团的正电荷并用中性脲基取代它。虽然瓜氨酸的生理效应尚不清楚，但已知这种PTM 调节许多过程，包括NET形成和细胞分化。 Coonrod小组最近的一份报告将组蛋白H3 R26鉴定为多种细胞系中PAD 2的体内靶点。该部位的瓜氨酸通过17 <$-雌二醇（E2）刺激雌激素受体<$$>（ER <$）而促进。下游效应包括染色质去凝聚和ER调控下200多个基因的转录激活（1）。有趣的是，与PAD 4不同，PAD 2不包含典型的核定位信号（NLS，56-PPAKKKST-63），我们假设蛋白质-蛋白质相互作用促进了向核的转运。PAD 2的晶体结构显示了几个潜在的对接位点，包括两个N-末端免疫球蛋白样结构域和下文所述的苯丙氨酸（FF）指基序（2）。目的1关注独特的FF基序，因为初步数据表明它通过充当未鉴定的调节蛋白的对接位点来调节PAD 2的细胞活性。为了解决这一假设，我们将采取一种组合和综合的方法，涉及生物化学和蛋白质组学实验，旨在确定这些新的PBPs。 在目标2中，我们将讨论第二个假设，其重点是瓜氨酸蛋白的“读者”存在的概念，以解释瓜氨酸蛋白并促进对瓜氨酸的生理反应。总之，这些研究将增加我们对调节PAD活性的机制的理解。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，从而增强我们对瓜氨酸蛋白生理作用的理解。我们还希望这些研究将有助于确定 精确的分子结构的酶，应该被靶向抑制，从而促进未来的努力，以开发化学探针和治疗靶向这些酶。

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.