Project 3: Targeting Pro-fibrotic E3 Ligases in Systemic Sclerosis

摘要项目#3：SSc中的泛素E3连接酶 系统性硬化症（SSc）是一种进行性多器官系统纤维化疾病， 自身免疫SSC相关死亡的主要原因是肺动脉高压（PAH）， 间质性肺病（ILD）。PAH影响10 - 15%的SSc患者。ILD的患病率各不相同 从40%到65%。SSc-ILD是CORT项目#3的主题。常见于许多 SSc疾病表型，包括皮肤和肺，是观察到成纤维细胞， 纤维化的主要效应细胞，呈现收缩性肌成纤维细胞表型，合成基质， 并表现出病态的寿命延长。发现新基因的传统方法 参与肺纤维化发病机制的研究已经进行了全基因组筛查， 揭示可能参与疾病发病机制的失调RNA种类。然而，在这方面， 这种方法可能会错过蛋白质的关键作用，这些蛋白质在蛋白质水平上不受调节。 转录，而是通过蛋白质降解，特别是通过泛素-蛋白酶体 泛素E3酶介导的UPS系统。我们最近的研究使用了大量的 特发性肺纤维化（IPF）和SSc肺样品已经揭示了一系列几种， 以前未被怀疑的分子目标。特别是，我们已经确定了两个高度新颖的亲- SSc成纤维细胞的纤维化信号通路：首先是新的胶原1基因阻遏物E2的丢失 转录因子8（E2 F8）通过遍在蛋白E3连接酶Fbxo16。这最终会导致 SSc成纤维细胞中的胶原合成。第二种是一种新的蛋白质亚型，称为FIEL1（纤维化- 诱导E3连接酶1），其有效刺激中枢促纤维化细胞因子TGF β信号传导 通过TGF β抑制剂PIAS 4的位点特异性泛素化和降解途径。此外，本发明还 我们已经开发出一种针对FIEL1的一流小分子抑制剂， 改善小鼠的纤维化。因此，我们假设E3成员的失调 泛素连接酶系统驱动SSc中的纤维化表型。在这个项目中，我们建议 筛选SSc-ILD肺和来源于SSc-ILD肺的肺成纤维细胞，以检测SSc-ILD肺的 SCF-和HECT-结构域泛素E3连接酶的成员，并测定它们在介导 病理性肌成纤维细胞表型。然后，我们将针对这些E3设计小分子抑制剂， 连接酶，并测试它们在肺纤维化动物模型中的有效性，以及我们独特的离体 患病的人肺灌注和培养系统。这些 拟议的研究将帮助我们发现一组新的潜在的药物靶点， SSc中纤维化途径的病理生物学。这些研究将提供一个基础平台， SSc-ILD中独特且具有潜在变革性的举措。我们的项目将与 其他CORT项目和核心，以发现泛素E3失调的重叠生物学 SSc-PAH和SSc-皮肤病中的连接酶。

ABSTRACT Project #3: Ubiquitin E3 ligases in SSc Systemic sclerosis (SSc) is a progressive multi-organ system fibrotic disorder associated with autoimmunity. The leading causes of SSc-related death are pulmonary hypertension (PAH) and interstitial lung disease (ILD). PAH affects 10-15% of SSc patients. The prevalence of ILD varies from 40 to 65% in cohort studies. SSc-ILD is the subject of CORT Project #3. Common to many of the SSc disease phenotypes, include the skin and the lung, is the observation that fibroblasts, the principal effector cells of fibrosis, assume the contractile myofibroblast phenotype, synthesize matrix, and exhibit a pathologically increased lifespan. A traditional approach to discover novel genes involved in the pathogenesis of pulmonary fibrosis has been to perform genome-wide screening to uncover dysregulated RNA species that may be involved in the pathogenesis of disease. However, this approach may miss the critical role of proteins that are NOT regulated at the level of transcription but rather through protein degradation, particularly through the ubiquitin-proteasome system (UPS) mediated by ubiquitin E3 enzymes. Our recent studies using a large number of idiopathic pulmonary fibrosis (IPF) and SSc lung samples have uncovered an array of several, previously unsuspected, molecular targets. In particular, we have identified two highly novel pro- fibrotic signaling pathways in SSc fibroblasts: First is the loss of a new collagen 1 gene repressor, E2 transcriptional factor 8 (E2F8) by a ubiquitin E3 ligase, Fbxo16. This ultimately leads to increased collagen synthesis in SSc fibroblasts. The second is a new protein isoform, termed FIEL1 (Fibrosis- Inducing E3 Ligase 1), which potently stimulates the central pro-fibrotic cytokine, TGFβ, signaling pathway through the site-specific ubiquitination and degradation of the TGFβ inhibitor PIAS4. Further, we have developed a first-in-class small molecule inhibitor towards FIEL1 that is highly effective in ameliorating fibrosis in mice. Thus, we hypothesize that dysregulation of members of E3 ubiquitin ligase system drives the fibrotic phenotype in SSc. In this project, we propose to screen SSc-ILD lungs and lung fibroblasts derived from SSc-ILD lungs for dysregulated expression of members of the SCF- and HECT-domain ubiquitin E3 ligases and to assay their function in mediating the pathologic myofibroblast phenotype. We will then design small molecule inhibitors for these E3 ligases and test them for effectiveness in animal models of lung fibrosis as well as our unique ex vivo diseased human lung perfusion and culture systems here at the University of Pittsburgh. These proposed studies will help us discover a new set of potentially druggable targets underlying the pathobiology of fibrotic pathways in SSc. These studies will provide a fundamental platform for a unique and potentially transformative initiative in SSc-ILD. Our project will interact very closely with the other CORT projects and cores to discover the overlapping biology of dysregulated ubiquitin E3 ligases in SSc-PAH and SSc-skin disease.