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

抑制人角膜细胞的细胞死亡：非手术圆锥角膜治疗的新见解

• 批准号: 10720431
• 负责人: Xinyue Liu
• 金额: $ 12.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10720431
• 关键词: 3-Dimensional; Advisory Committees; Affect; Affinity; American; Antibodies; Apoptosis; Apoptotic; Binding; Biological Assay; Biology; CASP8 gene; Caspase; Caspase Inhibitor; Cell Death; Cell Line; Cell Surface Receptors; Cell model; Cell surface; Cells; Cellular Stress; Cellular biology; Cessation of life; Collagen; Cone; Consumption; Contact Lenses; Cornea; Corneal Diseases; Corneal dystrophy; Coupled; Cysteine; DNA Damage; Data; Development; Disease; Dose; Drug Screening; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Fibroblasts; Glycosaminoglycans; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; Image; In Vitro; Inflammation; Inhibition of Apoptosis; Interruption; Keratoconus; Keratoplasty; Kinetics; Knowledge; Lead; Length; Ligands; Mass Spectrum Analysis; Mentors; Microarray Analysis; Modeling; NMR Spectroscopy; Nuclear Magnetic Resonance; Oligosaccharides; Operative Surgical Procedures; Pathology; Patients; Pattern; Phase; Phenotype; Phosphorylation; Play; Polymers; Polysaccharides; Proteins; Proteoglycan; Proteome; Proteomics; Recovery; Research; Research Personnel; Resources; Role; Sampling; Shapes; Signal Transduction; Stimulus; Stress; Stromal Cells; Structure; Sulfate; Surface; TNF gene; Thinness; Toxic effect; Training; Transplantation Surgery; Tumor Necrosis Factor Receptor; Tumor Promotion; Visualization; advanced analytics; biomacromolecule; career development; cell killing; crosslink; cytokine; dimer; drug discovery; extracellular; high throughput technology; improved; inhibitor; insight; instrumentation; medical schools; novel; polymerization; preference; prevent; protective effect; receptor binding; recruit; response; screening; skills; small molecule; targeted treatment; therapy design

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.

项目概要/摘要 圆锥角膜 (KC) 是一种进行性角膜疾病，每 500 至 2000 名美国人中就有一人患有圆锥角膜 圆锥形角膜图像扭曲 10-20% 的 KC 患者最终需要进行角膜移植。 角膜细胞凋亡和角膜胶原变性导致基质变薄，从而导致角膜基质变薄。 迄今为止，还没有针对角膜细胞凋亡的治疗方法。 寻找通过抑制角膜细胞外源性凋亡来治疗 KC 病例的非手术治疗方法 细胞外应激。人类肿瘤坏死因子 - α (TNF-α)，一种细胞因子释放的细胞因子。 炎症反应，在硫酸乙酰肝素（HS）的帮助下结合其细胞表面受体 蛋白多糖 (PG) 与 TNF-α 结合后，TNF 受体将开始在细胞内募集蛋白质， 这会导致 procaspase-8 二聚体的自我裂解和 caspase-8 (CASP8) 的激活。 裂解下游的执行半胱天冬酶并启动外源性细胞凋亡的级联反应。 该提案旨在寻找抑制激活的 CASP8 (AIM 1) 和 HS 的小分子化合物 抑制 TNF-α 和 TNF 受体之间相互作用的化合物 (AIM 3)。 抑制，我提出由样品多重质谱法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，以发现化合物可以干扰 TNF-α 和 HS。 TNF受体相互作用，我将利用聚糖微阵列技术筛选超过90种HS化合物 不同的硫酸化模式和链长度我将使用二维溶液核磁共振 (NMR)。 光谱学以获得相关蛋白质-配体相互作用的动力学 (AIM 1) 并验证圆锥角膜 3D-KC 细胞模型 (AIM 2&3) 中细胞凋亡抑制的影响。 该提案将受益于我的分析仪器技能，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） 我将获得 KC 生物学和病理学以及高级方面的进一步培训。 在指导的 K99 阶段，我将 1) 开发先进的管道。 针对圆锥角膜基质细胞凋亡的药物发现，2) 增强我在 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) 增强我的专业水平 成为一名成功的独立调查员所需的技能 该项目将在以下机构的咨询下进行。 我的导师 Steven Gygi 博士和我的顾问委员会：Dimitrios Karamichos 博士、刘玉涛博士、刘健博士、 哈佛医学院细胞生物学系也将提供极好的机会。 所提出的研究策略与职业发展培训相结合，将为我提供指导。 成为圆锥角膜生物学领域的独立研究者。