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.

项目摘要/摘要 角膜结构（KC）是一种进步的角膜疾病，每500至2000美国人中有一个 带有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，这 进一步诱导了KC。直到今天，还没有针对角膜细胞凋亡的治疗。该建议旨在 通过在角膜细胞中抑制外部凋亡，以搜索针对KC病例的非手术治疗 细胞外应激。人类肿瘤坏死因子 - α（TNF-α），一种在 炎症反应将在硫酸乙酰肝素（HS）的帮助下结合其细胞表面受体 蛋白聚糖（PG）。与TNF-α结合后，TNF受体将在细胞内启动蛋白质募集， 这导致了procaspase-8二聚体的自切除和caspase-8（CASP8）的激活。 CASP8会 清除下游的行政caspase，并启动一系列外部凋亡。 该建议旨在找到抑制激活的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）。完成CASP8 抑制作用，我提出了通过样品多重质谱法制备的无偏蛋白质组学方法 和基于抗体的凋亡标记。找到可以中断TNF-α和的HS化合物 TNF受体相互作用，我将利用聚糖微阵列技术来筛选90 hs的化合物 不同的硫酸化模式和链长。我将使用2D分辨率的核磁共振（NMR） 光谱学获得相关蛋白质 - 配体相互作用的动力学（AIM 1）并验证角膜菜谱 3D-KC细胞模型中凋亡抑制作用的影响（AIM 2＆3）。 该建议将受益于我的分析仪器技能，包括质谱和 溶液NMR，以及我对表征生物乳清分子的知识，包括蛋白质（蛋白质组学） 和糖胺聚糖（糖果）。我将获得KC生物学和病理学的进一步培训以及高级 药物筛查管道发展。在物质K99阶段，我将1）开发高级管道 靶向角质性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与糖基因之间的联系，4）增强我的专业人士 技能需要成为成功的独立研究者。该项目将根据 我的导师史蒂文·吉吉（Steven Gygi）博士和我的咨询委员会：Dimitrios Karamichos博士，Liu博士，Jian Liu博士，Jian Liu博士， 和Lianchun Wang。哈佛医学院细胞生物学系还将提供极好的机会 为了我的培训。拟议的研究策略加上职业发展培训，将指导我 成为圆锥角膜生物学领域的独立研究者。