PAD4 in Retinal Gliosis

瓜氨酸化是蛋白质翻译后修饰(PTM)引起的一种重要现象 通过将蛋白质上的精氨酸残基不可逆地修饰为非 由肽基精氨酸脱亚胺酶(PADS)编码的瓜氨酸残基。 瓜氨酸化是许多关键蛋白质的永久/不可逆转的变化 靶标和瓜氨酸蛋白已被检测到是疾病特异性的 几种主要炎症性疾病的生物标记物，如类风湿性关节炎， 多发性硬化症(MS)。带有小分子抑制剂的靶向垫 已被证明在几个临床前阶段有效地减轻了疾病负担 模特们。我们最近已经证明，瓜氨酸化是在 小鼠视网膜碱损伤。特别有趣的是，这一发现激活了 Muller胶质细胞和视网膜星形胶质细胞显示瓜氨酸可溶性水平升高 胶质纤维酸性蛋白(GFAP)。此外，瓜氨酸化的抑制会改变 可溶性GFAP的表达特征及其与视网膜的联系 神经胶质增多症。我们发现在瓜氨酸化途径中发现了这些增加 在严重碱损伤引起的慢性胶质细胞增多症期间，但在较温和的碱损伤中逆转 烧掉模型。有趣的是，激活的穆勒胶质细胞选择性地表达PAD4， 它负责GFAP的瓜氨酸化，PAD4的抑制减少 可溶性GFAP表达。在这份R21拨款提案中，我们将检验假设 PAD4驱动的瓜氨酸化控制激光中的慢性胶质细胞反应 老年性黄斑变性损伤模型的建立。在具体目标1中，我们 将研究PAD4在急性和慢性过程中的表达动态 胶质细胞增生症和PAD4‘S与瓜氨酸化和胶质纤维酸性蛋白的过度表达 S在不同的损伤范式中的修改。在目标2中，我们将调查 抑制PAD4酶活性和引起PAD4的后果 通过条件性靶基因敲除策略导致星形胶质细胞/胶质细胞缺陷， 并研究这种关键酶的抑制/缺失如何影响损伤 回应。这种关注PAD4的方法将有助于确定PAD4 在损伤状态下体内可用药，并首次奠定了基础 在导致不可逆转的AMD等条件下探索这种可用药的靶点 失明。

Citrullination is an important protein posttranslational modification (PTM) caused by an irreversible modification of the arginine residues on proteins to the non- coded citrulline residues by the peptidyl arginine deiminase enzymes (PADs). Citrullination is a permanent/irreversible change in numerous critical protein targets, and citrullinated proteins have been detected as disease-specific biomarkers in several major inflammatory diseases, such as rheumatoid arthritis, and multiple sclerosis (MS). Targeting PADs with small molecule inhibitors has been shown to be effective in reducing disease burden in several preclinical models. We have recently demonstrated that citrullination is induced in the mouse retina by alkali injury. Of particular interest is the discovery that activated Muller glia, and retinal astrocytes displayed elevated levels of citrullinated soluble glial fibrillary acidic protein (GFAP). Furthermore, inhibition of citrullination alters expression characteristics of soluble GFAP, and linked PAD's activity to retinal gliosis. We discovered that these increases in the citrullination pathway are found during chronic gliosis induced by severe alkali injury, but reversed in milder alkali burn model. Interestingly, activated Muller glia selectively expressed PAD4, which is responsible for GFAP citrullination, and inhibition of PAD4 reduces soluble GFAP expression. In this R21 grant proposal, we will test the hypothesis that PAD4-driven citrullination controls the chronic gliotic response in the laser injury model of age-related macular degeneration (AMD). In specific Aim 1, we will investigate the dynamics of PAD4 expression during acute versus chronic gliosis, and PAD4's association with overexpression of citrullination, and GFAP's modification(s) in different injury paradigms. In Aim 2, we will investigate the consequence of inhibiting PAD4 enzymatic activity as well as causing PAD4 deficiency in astrocytes/glial cells through conditional target knockout strategy, and investigate how the inhibition/absence of this critical enzyme affects injury response. This approach that focuses on PAD4 will help establish whether PAD4 is druggable in vivo in injury states and for the first time lay down the foundation to probe this druggable target in conditions, such as AMD, that cause irreversible blindness.