Prohibiting Cell Death in Human Keratocytes: New Insights for Non-surgical Keratoconus Treatment

项目摘要/摘要 圆锥角膜(KC)是一种进行性角膜疾病，每500到2000名美国人中就有一人看到 带有锥形角膜的扭曲图像。10%-20%的KC患者最终将需要角膜移植 手术。角膜中的角质细胞凋亡和胶原变性导致基质变薄，从而 进一步诱导KC。到目前为止，还没有针对角质细胞凋亡的治疗方法。这项建议旨在 通过抑制角质形成细胞外源性凋亡寻找治疗KC的非手术方法 细胞外压力。人肿瘤坏死因子-α是一种在血管内皮细胞中释放的细胞因子 炎症反应，将在硫酸肝素(HS)的协助下与其细胞表面受体结合 蛋白多糖(PG)。一旦与肿瘤坏死因子-α结合，肿瘤坏死因子受体将开始在细胞内募集蛋白质， 导致原天冬氨酸氨基转移酶-8二聚体的自我裂解和caspase-8(CASP8)的激活。Casp8将 切断执行半胱氨酸天冬氨酸酶下游，启动外源性细胞凋亡的级联反应。 这项建议旨在寻找抑制激活的CASP8(AIM 1)和HS的小分子化合物 抑制肿瘤坏死因子-α和肿瘤坏死因子受体之间相互作用的化合物(目标3)。完成CASP8 抑制，我提出了通过样品多路复用质谱学促进的无偏蛋白质组学方法 以及基于抗体的细胞凋亡标记物检测。寻找能够阻断肿瘤坏死因子-α和 肿瘤坏死因子受体相互作用，我将利用葡聚糖微阵列技术筛选90多个HS化合物 不同的硫酸盐化模式和链长。我将使用2D-溶液核磁共振(核磁共振) 获得相关蛋白质-配体相互作用的动力学的光谱(AIM 1)和验证圆锥角膜 凋亡抑制在3D-KC细胞模型中的作用(AIM 2和3)。 这项提议将受益于我的分析仪器技能，包括质谱学和 溶液核磁共振，以及我对包括蛋白质在内的生物大分子的表征知识(蛋白质组学) 和糖胺多聚糖(糖链)。我将获得KC生物学和病理学方面的进一步培训，并将 药物筛选流水线的开发。在K99指导阶段，我将1)开发一条先进的管道 针对圆锥角膜基质细胞凋亡的药物发现，2)加强我对KC生物学的了解，3) 围绕KC研究在蛋白质组学和糖组学之间建立联系，并4)提高我的专业水平 成为一名成功的独立调查员所需的技能。该项目将在以下顾问的建议下进行 我的导师Steven Gygi博士和我的顾问委员会：迪米特里奥斯·卡拉米霍斯博士、刘玉涛博士、刘健博士， 还有王连春。哈佛医学院细胞生物学系也将提供绝佳的机会 为了我的训练。提出的研究战略，结合职业发展培训，将指导我 成为圆锥角膜生物学领域的独立研究员。

Project Summary/Abstract Keratoconus (KC) is a progressing cornea disorder that causes one in every 500 to 2000 Americans to see distorted images with a cone-shaped cornea. 10-20% of KC patients will ultimately require cornea transplant surgeries. Keratocyte apoptosis and collagen degeneration in the cornea lead to stromal thinning, which further induces KC. Up to today, there is no therapy targeted to keratocyte apoptosis. This proposal aims to search for non-surgical treatments for KC cases by inhibiting extrinsic apoptotic in keratocytes under extracellular stress. The human tumor necrosis factor – alpha (TNF-α), a cytokine released in the inflammation response, will bind its cell surface receptors with the assistance of heparan sulfate (HS) proteoglycan (PG). Upon binding with TNF-α, the TNF receptors will start protein recruitment intracellularly, which leads to the self-cleavage of procaspase-8 dimer and the activation of caspase-8 (CASP8). CASP8 will cleave the executive caspases downstream and initiate the cascade of extrinsic apoptosis. This proposal aims to find small molecule compounds that inhibit activated CASP8 (AIM 1) and HS compounds that inhibit interactions between TNF-α and TNF receptors (AIM 3). To accomplish CASP8 inhibition, I propose unbiased proteomics approaches facilitated by sample multiplexing mass spectrometry and antibody-based assays for markers of apoptosis. To find HS compounds that can interrupt TNF-α and TNF receptors interaction, I will utilize glycan microarray technology to screen over 90 HS compounds with different sulfation patterns and chain lengths. I will use 2D-solution nuclear magnetic resonance (NMR) spectroscopy to obtain kinetics of associated protein-ligand interactions (AIM 1) and validate keratoconic effects of the apoptotic inhibitions in 3D-KC cell models (AIM 2&3). This proposal will benefit from my analytical instrumentation skills, including mass spectrometry and solution NMR, and my knowledge about characterizing biomacromolecules, including proteins (proteomics) and glycosaminoglycans (glycomics). I will obtain further training in KC biology and pathology and advanced drug screening pipeline development. During the mentored K99 phase, I will 1) develop an advanced pipeline of drug discovery targeting keratoconic stromal cell apoptosis, 2) strengthen my knowledge in KC biology, 3) build connections between proteomics and glycomics around KC research, and 4) enhance my professional skills needed to be a successful independent investigator. The project will be conducted under the advisory of my mentor Dr. Steven Gygi and my advisory committee: Dr. Dimitrios Karamichos, Dr. Yutao Liu, Dr. Jian Liu, and Lianchun Wang. Harvard Medical School Cell Biology Department will also provide excellent opportunities for my training. The proposed research strategy, combined with the career development training, will guide me to become an independent investigator in the field of Keratoconus biology.