Protein Arginine Deiminase 2 (PAD2) and Protein Citrullination in ALS

项目摘要 肌萎缩侧索硬化症(ALS)是一种导致运动神经元的进行性神经退行性疾病 退化、肌肉无力、瘫痪和死亡。运动神经元变性的机制是 目前还不完全了解，没有任何治疗方法可以阻止或逆转疾病的发展。至 了解该病，开辟治疗新途径，探索该病新的发病机制 需要的。为此，徐实验室和汤普森实验室联手研究蛋白质的作用 在ALS中，由精氨酸脱亚氨酶(PADS)蛋白催化的瓜氨酸化反应。瓜氨酸去除正性 来自蛋白质的电荷。因此，这种修饰可以改变蛋白质的功能及其与其他蛋白质的相互作用 蛋白质、膜和核酸。哺乳动物有五个垫：垫1-4和垫6。PAD2是 中枢神经系统(CNS)中的优势形式。先前的研究表明，PAD2和蛋白质 神经退行性疾病，如阿尔茨海默病和普里恩病，瓜氨酸化增加。 然而，还没有研究将PAD和蛋白质瓜氨酸化与ALS联系起来。此外，没有研究表明 研究了PAD2和蛋白质瓜氨酸化在神经退行性变中的功能作用。为了填补这些知识 GAP，徐和汤普森实验室应用了他们在肌萎缩侧索硬化症和蛋白质瓜氨酸化方面的组合专业知识和 发起了这次调查。我们的初步研究表明，明确的证据表明，PAD2的表达(但不是 在两个ALS小鼠模型中，PAD3，4)和蛋白质瓜氨酸化在空间和时间上都发生了变化 表达突变体SOD1G93A和表达突变体PFN1C71G。而PAD2的表达增加 在星形胶质细胞中，在疾病进展过程中，它在神经元中的表达减少。通过蛋白质组学，我们 在脊髓中鉴定出数百种瓜氨酸蛋白。在ALS小鼠中，疾病的早期阶段是 以蛋白质瓜氨酸化减少为主。相反，疾病晚期表现出瓜氨酸化增加。 在一半的蛋白质中，另一半的瓜氨酸化减少。耐人寻味的是，高度瓜氨酸 后期蛋白质主要集中在不溶组分中。我们建议通过以下方式进一步调查 回答以下七个问题：(1)在肌萎缩侧索硬化症中蛋白质瓜氨酸化是如何改变的？(2)什么是 脊髓中的瓜氨酸化蛋白，以及在ALS中这些蛋白中的瓜氨酸化是如何改变的？(3)什么是 肌萎缩侧索硬化症相关蛋白上的瓜氨酸化部位？(4)PAD2是否对改变的蛋白负责？ 肌萎缩侧索硬化症中的瓜氨酸化？(5)蛋白质瓜氨酸化如何影响蛋白质聚集？(6) 肌萎缩侧索硬化症相关靶点的瓜氨酸化影响其蛋白质功能？(7)蛋白瓜氨酸化如何改变 人类肌萎缩侧索硬化症？通过回答这些问题，我们将进入并调查一个迄今尚未探索的研究领域 从而为理解肌萎缩侧索硬化症的神经退变机制开辟了一个新的维度。 由于PAD抑制剂已经在汤普森实验室开发出来，这一新维度可能会导致 针对PAD2活性的新治疗途径。由于蛋白质瓜氨酸化的显著变化， 这项研究还可能识别早期ALS的新生物标记物。 1

Project Summary Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes motor neuron degeneration, muscle weakness, paralysis, and death. The mechanism of motor neuron degeneration is incompletely understood, and currently, no therapy can arrest or reverse the disease progression. To understand the disease and open new avenues for therapy, exploring new mechanisms of the disease is needed. To this end, the Xu and Thompson labs have joined forces to investigate the role of protein citrullination, which is catalyzed by Protein Arginine Deiminases (PADs), in ALS. Citrullination removes positive charges from proteins. Therefore, this modification can alter the protein function and its interaction with other proteins, membranes, and nucleic acids. Mammals have five PADs: PADs 1–4 and PAD6. PAD2 is the dominant form in the central nervous system (CNS). Previous studies have shown that PAD2 and protein citrullination are increased in neurodegenerative diseases such as Alzheimer's disease and Prion disease. However, no studies have linked PADs and protein citrullination with ALS. Furthermore, no studies have investigated the functional role of PAD2 and protein citrullination in neurodegeneration. To fill these knowledge gaps, the Xu and Thompson labs have applied their combined expertise in ALS and protein citrullination and initiated this investigation. Our preliminary studies show unequivocal evidence that PAD2 expression (but not PAD3, 4) and protein citrullination are spatially and temporally altered in two ALS mouse models, one expressing mutant SOD1G93A and the other expressing mutant PFN1C71G. While PAD2 expression is increased in astrocytes, its expression is decreased in neurons during the disease progression. By proteomics, we identified several hundred citrullinated proteins in the spinal cord. In ALS mice, the early disease stage is dominated by decreased protein citrullination. In contrast, the late disease stage shows increased citrullination in one-half of the proteins and decreased citrullination in the other half. Intriguingly, the highly citrullinated proteins are enriched in the insoluble fractions in the late stage. We propose to further our investigation by answering the following seven questions: (1) How is protein citrullination altered in ALS? (2) What are the citrullinated proteins in the spinal cord, and how is citrullination altered in these proteins in ALS? (3) What are the sites of citrullination on ALS-associated proteins? (4) Is PAD2 responsible for the altered protein citrullination in ALS? (5) How does protein citrullination impact protein aggregation? (6) How does the citrullination of ALS-associated targets impact their protein function? (7) How is protein citrullination altered in human ALS? By answering these questions, we will enter and investigate a hitherto unexplored research area in ALS, thereby opening a new dimension in understanding the mechanism of neurodegeneration in ALS. Because PAD inhibitors have already been developed in the Thompson lab, this new dimension may lead to new therapeutic avenues targeting PAD2 activity. Because of the significant changes in protein citrullination, this research may also identify novel biomarkers of early-stage ALS. 1