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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

基本信息

批准号：
10321541
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
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10321541
关键词：
Active Sites Affect Age Alzheimer&apos s Disease Antibodies Arabidopsis Aspartic Endopeptidases Binding Caenorhabditis elegans Cathepsins 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

项目摘要

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，但仍不清楚颗粒蛋白前体缺乏如何导致额颞叶变性（FTLD）并导致阿尔茨海默病（AD）。颗粒蛋白原是裂解成功能上对抗颗粒蛋白前体全蛋白的生物活性颗粒蛋白肽。许多人认为颗粒蛋白前体单倍不足同样会消耗颗粒蛋白前体和颗粒蛋白水平，但这从未被直接测量。关于将颗粒蛋白前体切割成产生颗粒蛋白，这些裂解肽的正常功能和颗粒蛋白对蛋白质稳态和疾病。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影响神经退行性疾病如AD和FTLD的风险。客观本申请的目的是利用C. 线虫、培养细胞和患者来源的生物样本。核心假设是年龄和生理应激促进颗粒蛋白在内溶酶体系统中的释放，颗粒蛋白在内溶酶体系统中结合并抑制特定的溶酶体蛋白酶，最终损害神经元蛋白质稳态。这假设是基于申请人产生的大量初步数据，随着年龄和胁迫的增加，颗粒蛋白促进TDP-43的积累并增强其毒性，富含半胱氨酸的肽可以结合并在空间上抑制溶酶体N-乙酰基蛋白酶的活性位点，组织蛋白酶D这项工作的基本原理是，了解颗粒蛋白前体的产生和功能，而颗粒蛋白对于在FTLD和其他疾病的治疗中安全地靶向这些分子至关重要。中心假设将通过三个具体目标进行测试：1）了解受管制的生产，颗粒蛋白，2）确定颗粒蛋白抑制组织蛋白酶的特异性谱，和3）阐明颗粒蛋白对蛋白质稳态的影响。这项研究在概念上是创新的，因为它试图直接暗示与年龄相关的颗粒蛋白积累，而不是或除了颗粒蛋白前体缺乏，作为与颗粒蛋白前体突变相关的神经变性的驱动力。从技术上讲，由于开发了一种新的荧光颗粒蛋白前体裂解传感器工具，一组抗人颗粒蛋白抗体和生物层干涉法（BLI）在无标记、真实的- 颗粒蛋白/组织蛋白酶相互作用动力学的时间监测。这项研究将有助于至关重要的关于颗粒蛋白的生产、特异性和功能性结果的信息。这种贡献重要的是因为它将提高对颗粒蛋白前体单倍不足如何导致FTLD的理解，提供了关于溶酶体蛋白酶正常调节的基本新知识，为开发安全的颗粒蛋白前体替代疗法奠定了基础。