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

项目概要/摘要 圆锥角膜（KC）是一种进展性角膜疾病，导致每500至2000名美国人中就有一人失明。 圆锥形角膜的扭曲图像。10-20%的KC患者最终需要角膜移植 手术。角膜中的角膜细胞凋亡和胶原变性导致基质变薄， 进一步诱导KC。迄今为止，还没有针对角膜基质细胞凋亡的治疗方法。这项建议旨在 通过抑制角膜上皮细胞的外源性凋亡来寻找非手术治疗KC的方法 细胞外应激人肿瘤坏死因子-α（TNF-α），一种在肿瘤细胞中释放的细胞因子， 炎症反应，将结合其细胞表面受体的协助下，硫酸乙酰肝素（HS） 蛋白聚糖（PG）。与TNF-α结合后，TNF受体将开始细胞内蛋白质募集， 其导致半胱天冬酶-8二聚体的自切割和半胱天冬酶-8（CASP 8）的活化。CASP 8将 切割下游的执行性半胱天冬酶并启动外源性凋亡的级联反应。 该提案旨在寻找抑制活化的CASP 8（AIM 1）和HS的小分子化合物， 抑制TNF-α和TNF受体（AIM 3）之间相互作用的化合物。完成CASP 8 抑制，我提出了无偏见的蛋白质组学方法促进样品多路复用质谱 和基于抗体的凋亡标记物测定。为了找到可以阻断TNF-α和TNF-α分泌的HS化合物， TNF受体相互作用，我将利用聚糖微阵列技术筛选90多个HS化合物， 不同的硫酸化模式和链长。我将使用二维溶液核磁共振（NMR） 光谱学以获得相关蛋白-配体相互作用（AIM 1）的动力学并验证圆锥角膜 在3D-KC细胞模型（AIM 2&3）中的凋亡抑制作用。 这个建议将受益于我的分析仪器的技能，包括质谱和 溶液核磁共振，以及我关于表征生物大分子（包括蛋白质）的知识（蛋白质组学） 和糖胺聚糖（糖组学）。我将获得进一步的培训，在KC生物学和病理学和先进的 药物筛选管道开发。在辅导K99阶段，我将1）开发一个先进的管道 2）加强我在KC生物学方面的知识，3） 围绕KC研究建立蛋白质组学和糖组学之间的联系，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。该项目将在咨询下进行， 我的导师Steven Gygi博士和我的顾问委员会：Dimitrios Karamichos博士，Yutao Liu博士，Jian Liu博士， 和王连春。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策略，结合职业发展培训，将对我起到指导作用 成为圆锥角膜生物学领域的独立研究者。

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.