Deregulation of the Ubiquitin/26S Proteasome Pathway in AT cells: Role of ISG15

描述（由申请人提供）：共济失调毛细血管扩张症（A-T）（Boder-Sedgwick/Louis-Bar综合征）是一种遗传性免疫缺陷疾病，患病率为1/30，000 - 100，000新生儿。A-T患者的特征是明显的面部蜘蛛静脉（毛细血管扩张），复发性鼻窦炎感染和不规则门（共济失调），这是由进行性神经元功能障碍引起的。这些临床表现继发于对电离辐射的敏感性和明显的癌症易感性，Savitsky等人于1995年将其解释为共济失调毛细血管扩张症（ATM）基因的常染色体隐性突变。ATM是一种370 kDa的核Ser/Thr激酶，有助于调节p53、BRCA 1和Chk 2信号通路等，因此是通过有丝分裂检查点、双链DNA修复、端粒修复、凋亡和减数分裂进行所需的。ATM在细胞调控中的核心作用表明ATM基因的功能缺失突变会导致广泛的多效性效应。在初步研究中，我们已经表明靶向蛋白酶体介导的降解在A-T细胞中受损，这表明A-T细胞中蛋白质周转的减少可能有助于A-T表型（例如神经变性）。我们的初步研究还表明，A-T细胞中蛋白质周转的减少与ISG 15（干扰素刺激基因15）的表达升高有关，ISG 15是一种被证明拮抗泛素途径的泛素样蛋白。本申请由三个特定目的组成，其被设计用于探测ISG 15在共济失调毛细血管扩张症中的作用。在第一个具体目标中，我们将使用各种AT细胞系研究ATM在调节ISG 15表达中的作用。在第二个具体目标中，我们将测试ISG 15的表达升高是否损害A-T神经元中的蛋白质周转。最后，在第三个具体目标中，我们将研究ISG 15介导的泛素依赖性蛋白质降解缺陷是否在体外（使用培养中生长的神经元）和体内（Atm -/-小鼠）触发神经元细胞死亡。AT患者进行性神经变性的原因尚不清楚。从所提出的实验产生的结果将提供关于ATM/ISG 15在AT的神经变性中的作用的见解。未来可以开发针对ISG 15通路的小分子抑制剂，以预防AT患者中与神经变性相关的共济失调。因此，这些研究具有治疗A-T患者的潜在临床应用。公共卫生相关性：我们计划测试是否泛素样蛋白ISG 15的表达升高是负责共济失调毛细血管扩张症（A-T）的神经变性。本提案中描述的原理验证实验将为未来开发ISG 15通路的小分子抑制剂奠定基础。这些小分子抑制剂有望预防A-T患者的共济失调。

DESCRIPTION (provided by applicant): Ataxia Telangiectasia (A-T) (Boder-Sedgwick/Louis-Bar syndrome) is an inherited immunodeficiency disorder with a prevalence of 1 in 30,000-100,000 births. A-T patients are characterized by pronounced facial spider veins (telangiectsia), recurrent sinopulmonary infections, and an irregular gate (ataxia), which results from progressive neuronal dysfunctions. These clinical presentations, secondary to the sensitivity to ionizing radiation and a marked predisposition to cancer, were explicated in 1995 by Savitsky et al. as an autosomal recessive mutation in the Ataxia Telangiectasia (ATM) gene. ATM is a 370 kDa nuclear Ser/Thr kinase contributing to the regulation of p53, BRCA1, and Chk2 signaling pathways, amongst others, and thus required for progression through mitotic checkpoints, double strand DNA repair, telomere repair, apoptosis, and meiosis. The central role of ATM in cell regulation suggests that loss of function mutations in ATM gene should result in broad pleiotropic effects. In preliminary studies, we have shown that targeted proteasome-mediated degradation is impaired in A-T cells, suggesting that reduce protein turnover in A-T cells could potentially contribute to the A-T phenotype (e.g. neurodegeneration). Our preliminary studies also demonstrated that the reduced protein turnover in A-T cells is associated with elevated expression of ISG15, the Interferon- Stimulated Gene 15, an ubiquitin-like protein proven to antagonize ubiquitin pathway. The present application is comprised of three Specific Aims designed to probe the role of ISG15 in Ataxia Telangiectasia. In the first specific aim, we will investigate the role of ATM in regulating ISG15 expression using various AT cell lines. In the second specific aim, we will test if the elevated expression of ISG15 impairs protein turnover in A-T neurons. Finally, in the third specific aim, we will examine if ISG15-mediated defect in ubiquitin-dependent protein degradation triggers neuronal cell death in vitro (using neurons grown in culture) and in vivo (in Atm -/- mice). The reason(s) for the progressive neurodegeneration in AT patients is not known. Results generated from the proposed experiments will provide insights in to the role of ATM/ISG15 in neurodegeneration in AT. The small molecule inhibitors targeting ISG15 pathway could then be developed in future to prevent ataxia associated with neurodegeneration in AT patients. Thus these studies have potential clinical applications for treating A-T patients. PUBLIC HEALTH RELEVANCE: We plan to test if the elevated expression of ubiquitin-like protein ISG15 is responsible for the neurodegeneration in Ataxia Telangiectasia (A-T). The proof-of-principle experiments described in this proposal will form the foundation for future development of small molecule inhibitors for the ISG15 pathway. These small molecule inhibitors are expected to prevent the ataxia in A-T patients.