Understanding cleaved granulin production, protease inhibition and effects on protein homeostasis

了解裂解颗粒蛋白的产生、蛋白酶抑制以及对蛋白质稳态的影响

• 批准号: 10088360
• 负责人: Aimee Kao
• 金额: $ 35.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10088360
• 关键词: Active Sites; Affect; Age; Alzheimer' s Disease; Antibodies; Arabidopsis; Aspartic Endopeptidases; Binding; Caenorhabditis elegans; Cathepsins; Cleaved cell; Cultured Cells; Cysteine; Data; Dementia; Development; Disease; Foundations; Frontotemporal Dementia; Frontotemporal Lobar Degenerations; Glycoproteins; Goals; Human; Impairment; In Vitro; Inflammation; Interferometry; Kinetics; Knowledge; Label; Lead; Length; Life; Lysosomal Storage Diseases; Lysosomes; Measures; Mission; Modeling; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; PGRN gene; Parkinson Disease; Pathogenesis; Patients; Peptide Hydrolases; Peptides; Phylogeny; Physiological; Play; Population; Process; Production; Public Health; Regulation; Replacement Therapy; Research; Risk; Role; Specificity; Stress; System; Testing; Toxic effect; United States National Institutes of Health; Work; age related; base; biological adaptation to stress; burden of illness; driving force; granulin; granulin 2; granulin 3; improved; innovation; loss of function mutation; mutation carrier; neglect; new therapeutic target; novel; pleiotropism; protein TDP-43; proteostasis; real time monitoring; sensor; tool; wound healing

Despite recent progress, it remains unclear how progranulin deficiency leads to development of frontotemporal lobar degeneration (FTLD) and contributes to Alzheimer’s Disease (AD). Progranulin is cleaved into bioactive granulin peptides that functionally oppose the progranulin holoprotein. Many believe that progranulin haploinsufficiency equally depletes progranulin and granulin levels, yet this has never been directly measured. Fundamental knowledge gaps also exist regarding proteases that cleave progranulin to produce granulins, the normal function of these cleaved peptides and consequences of granulins on protein homeostasis and disease. The long-term goal of this research is to understand how age-related changes in protein homeostasis affect risk of neurodegenerative diseases like AD and FTLD. The objective of this application is to understand the production, function and consequences of granulins using C. elegans, cultured cells and patient-derived biospecimens. The central hypothesis is that age and physiological stress promote the liberation of granulins in the endolysosomal system, where granulins bind to and inhibit specific lysosomal proteases, ultimately impairing neuronal protein homeostasis. This hypothesis is based on extensive preliminary data produced by the applicant that granulin production increases with age and stress, granulins promote TDP-43 accumulation and enhance its toxicity, and these cysteine-rich peptides can bind and sterically inhibit the active site of a lysosomal aspartyl protease, cathepsin D. The rationale for this work is that understanding production and function of both progranulin and granulins is critical to safely targeting these molecules in the treatment of FTLD and other diseases. The central hypothesis will be tested through three specific aims: 1) understand the regulated production of granulins, 2) determine the specificity profile of cathepsin inhibition by granulins, and 3) elucidate the effects of granulins on protein homeostasis. The proposed research is conceptually innovative because it seeks to directly implicate age-associated granulin accumulation, rather than or in addition to progranulin deficiency, as a driving force in neurodegeneration related to progranulin mutations. It is also technically innovative because of development of a novel fluorescent progranulin cleavage sensor tool, use of a new set of anti-human granulin antibodies and application of BioLayer Interferometry (BLI) for label-free, real- time monitoring of granulin/cathepsin interaction kinetics. The proposed research will contribute essential information about the production, specificity and functional consequences of granulins. This contribution is significant because it will improve understanding of how progranulin haploinsufficiency leads to FTLD, provide fundamental new knowledge regarding the normal regulation of lysosomal proteases and lay the foundation for development of safe progranulin replacement therapies.

尽管最近取得了进展，但仍不清楚原颗粒缺乏是如何导致 额颞叶变性(FTLD)并导致阿尔茨海默病(AD)。原颗粒剂是 被切割成具有生物活性的颗粒多肽，在功能上对抗原颗粒全蛋白。许多人认为 原颗粒单倍体不足同样会消耗原颗粒和颗粒水平，但这从来都不是 直接测量的。关于将原颗粒蛋白裂解为 产生颗粒蛋白，这些被切割的多肽的正常功能以及颗粒蛋白对 蛋白质动态平衡与疾病。这项研究的长期目标是了解与年龄有关的 蛋白质稳态的变化会影响阿尔茨海默病和FTLD等神经退行性疾病的风险。目标是 这个应用的目的是用C语言理解颗粒剂的产生、功能和后果。 线虫、培养细胞和病人衍生的生物标本。中心假设是年龄和 生理应激促进内溶酶体系统中颗粒的释放，颗粒结合在这里 抑制特定的溶酶体蛋白水解酶，最终破坏神经元蛋白质的稳态。这 假设是基于申请者提供的大量初步数据，即颗粒蛋白生产 随着年龄和应激的增加，颗粒蛋白促进TDP-43的积聚并增强其毒性，这些 富含半胱氨酸的多肽可以结合和立体地抑制溶酶体天冬氨酸蛋白酶的活性部位， 组织蛋白D。这项工作的基本原理是理解两种原颗粒的产生和功能 在治疗FTLD和其他疾病时，颗粒素对于安全地靶向这些分子至关重要。 核心假设将通过三个具体目标进行检验：1)了解受监管的生产 颗粒蛋白，2)确定颗粒抑制组织蛋白酶的特异性，以及3)阐明 颗粒剂对蛋白质稳态的影响。拟议的研究在概念上是创新的，因为它 寻求直接涉及与年龄相关的颗粒蛋白积聚，而不是或除了PROGROING之外 缺乏，作为与原颗粒突变相关的神经退化的驱动力。它在技术上也是 创新是因为开发了一种新型的荧光蛋白裂解传感器工具，使用了一种新的 一组抗人颗粒蛋白抗体及其在生物层干涉法(BLI)中的应用 颗粒蛋白/组织蛋白酶相互作用动力学的时间监测。拟议的研究将对 有关颗粒剂的生产、特异性和功能后果的信息。这一贡献是 因为这将提高对原颗粒单倍体不足如何导致FTLD的理解， 提供有关溶酶体蛋白水解酶正常调节的基本新知识，并奠定 开发安全的原颗粒替代疗法的基础。