CTGF-interacting proteins in scleroderma lung fibrosis

描述(由申请人提供)：本提案为候选人在硬皮病研究方面的职业生涯奠定了基础。她接受的正式博士培训主要是药理学和受体信号传递。她对蛋白质组学技术产生了兴趣，并希望将其应用于硬皮病研究。在此授课期间，候选人希望通过教学和实验室经验，在风湿学、硬皮病成纤维细胞生物学、质谱学和蛋白质组学等领域培养急需的技能。然后，利用这些新技术，结合她的受体药理学和信号转导背景，她将开展独立的研究项目，研究硬皮病纤维化的基本机制。为此，已经准备了一份提案，该提案应该提供充分的机会来开发新的技能，并获得候选人长期目标所需的硬皮病研究的理论背景。 该提案的研究计划是基于观察到结缔组织生长因子(CTGF)与硬皮病患者肺成纤维细胞中的许多蛋白质特异性结合。CTGF在许多纤维化疾病中高表达，被认为是纤维化的分子标志物。间质性肺纤维化影响了高达80%的硬皮病患者，现在是导致死亡的主要原因。由于硬皮病肺成纤维细胞不仅合成增加的CTGF，而且还含有与CTGF特异相互作用的蛋白质，我们推测这种相互作用在硬皮病肺纤维化的发生和发展中起关键作用。我们的研究计划旨在鉴定CTGF相互作用的蛋白，并确定硬皮病肺成纤维细胞中CTGF信号转导复合体的组成。 该项目将结合生物化学和蛋白质组学技术，在硬皮病肺成纤维细胞中详细重建CTGF的微环境。拟议的部分研究将使用Biacore 3000进行，它允许检测蛋白质的瞬时结合。以这种方式使用这种商业工具是一种新的技术，这种技术并不常见。使这一建议特别独特的另一个因素是设备齐全的质谱分析设备的可用性，该设备提供了在皮摩尔水平或更低水平对蛋白质进行测序的仪器。尖端的蛋白质组学技术将使我们能够识别通过ID或2D凝胶电泳解析的或从Biacore传感器芯片洗脱出来的CTGF相互作用的蛋白质，以及发现与CTGF交叉连接的蛋白质复合体。已确定的蛋白(S)可能成为治疗硬皮病肺纤维化的潜在靶点(S)，目前尚无有效的治疗方法。

DESCRIPTION (provided by applicant): This proposal provides a means of laying the groundwork for the candidate's career in scleroderma research. Her formal Ph.D. training was primarily in pharmacology and receptor signaling. She has developed an interest in proteomics technologies and wants to apply them to scleroderma research. In the course of this award period, the candidate expects to develop skills in the areas of rheumatology, biology of scleroderma fibroblasts, mass spectrometry, and proteomics that are much needed, through both didactic and laboratory experience. Then, using these new skills in combination with her receptor pharmacology and signal transduction background, she will develop independent research projects investigating the fundamental mechanisms of scleroderma fibrosis. Toward these ends, a proposal has been prepared which should provide ample opportunity to develop new skills and to obtain the theoretical background in scleroderma research required for the candidate's long-term goals. The research plan of the proposal is based on the observations that connective tissue growth factor (CTGF) Specifically binds numerous proteins in lung fibroblasts derived from scleroderma patients. CTGF is highly expressed in many fibrotic disorders and is considered to be a molecular marker of fibrosis. Interstitial lung fibrosis strikes up to 80% of scleroderma patients and is now the leading cause of death. Since scleroderma lung fibroblasts not only synthesize increased amounts of CTGF, but also contain proteins that specifically interact with CTGF, we postulate that such interactions are critical for the pathogenesis and expansion of scleroderma pulmonary fibrosis. Our research plan aims to identify the CTGF-interacting proteins and to define the components of a signal transduction complex for CTGF in scleroderma lung fibroblasts. This project will involve a combination of biochemical and proteomic techniques applied to the detailed reconstruction of CTGF's microenvironment in scleroderma lung fibroblasts. Part of the proposed studies will be carried out with BIAcore 3000, which allows detection of even transient binding of proteins. The use of this commercial instrument in this manner is a novel technique, which is not commonly available. An additional factor that makes this proposal particularly unique is the availability of the well-equipped Mass Spectrometry Facility, which offers instrumentation to sequence proteins at picomolar levels and lower. Cutting-edge proteomics technologies will allow us to identify CTGF-interacting proteins resolved via ID or 2D gel electrophoresis or eluted from the BIAcore sensor chip, as well as to spot the complex of protein cross linked to CTGF. The identified protein(s) might serve then as potential target(s) in the treatment of scleroderma pulmonary fibrosis for which no proven, effective therapy currently exists.