蛋白前蛋白缺失神经元中溶酶体功能和蛋白稳态的多模态蛋白质组学特征。

IF 14.9 1区 医学 Q1 NEUROSCIENCES
Saadia Hasan, Michael S Fernandopulle, Stewart W Humble, Ashley M Frankenfield, Haorong Li, Ryan Prestil, Kory R Johnson, Brent J Ryan, Richard Wade-Martins, Michael E Ward, Ling Hao
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引用次数: 0

摘要

背景:前颗粒蛋白(PGRN)是一种溶酶体糖蛋白,与多种神经退行性疾病有关,包括额颞叶痴呆和神经性ceroid脂褐质病。在GRN基因中发现的超过70个突变都导致PGRN蛋白的表达减少。遗传和功能研究指出PGRN在溶酶体功能中的调节作用。然而,PGRN在溶酶体中的详细分子功能以及PGRN缺乏对溶酶体的影响尚不清楚。方法:我们开发了多方面的蛋白质组学技术来表征活的人类神经元和固定的小鼠脑组织中的动态溶酶体生物学。利用溶酶体接近标记和完整溶酶体的免疫纯化,我们对人类诱导多能干细胞(iPSC)衍生的谷氨酸能神经元(i3Neurons)和小鼠大脑中的溶酶体组成和相互作用组进行了表征。利用细胞培养氨基酸动态稳定同位素标记(dSILAC)蛋白质组学,我们首次测量了人类i3神经元的整体蛋白质半衰期。结果:利用多模态蛋白质组学和活细胞成像技术,我们全面表征了PGRN缺乏如何改变神经元溶酶体的分子和功能景观。我们发现,PGRN缺失损害了溶酶体的降解能力,溶酶体膜上v-ATPase亚基水平升高,溶酶体内水解酶增加,与溶酶体运输相关的蛋白质调节改变,溶酶体ph升高。与溶酶体功能受损一致,GRN缺失的i3神经元和携带GRN突变的额颞叶痴呆患者来源的i3神经元的蛋白质周转明显改变。如组织蛋白酶和与超分子聚合和遗传性神经退行性疾病相关的蛋白质。结论:本研究提示PGRN是溶酶体pH值和降解能力的关键调节因子,影响神经元的整体蛋白质稳态。除了对颗粒蛋白前缺失的研究外,这些新发展的神经元和脑组织蛋白质组学方法为研究高动态的神经元溶酶体生物学提供了有用的工具和数据资源。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons.

Background: Progranulin (PGRN) is a lysosomal glycoprotein implicated in various neurodegenerative diseases, including frontotemporal dementia and neuronal ceroid lipofuscinosis. Over 70 mutations discovered in the GRN gene all result in reduced expression of the PGRN protein. Genetic and functional studies point toward a regulatory role for PGRN in lysosome functions. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomes remain unclear.

Methods: We developed multifaceted proteomic techniques to characterize the dynamic lysosomal biology in living human neurons and fixed mouse brain tissues. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactome in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in human i3Neurons for the first time.

Results: Leveraging the multi-modal proteomics and live-cell imaging techniques, we comprehensively characterized how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. We found that PGRN loss impairs the lysosome's degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased hydrolases within the lysosome, altered protein regulations related to lysosomal transport, and elevated lysosomal pH. Consistent with impairments in lysosomal function, GRN-null i3Neurons and frontotemporal dementia patient-derived i3Neurons carrying GRN mutation showed pronounced alterations in protein turnover, such as cathepsins and proteins related to supramolecular polymerization and inherited neurodegenerative diseases.

Conclusion: This study suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which influences global proteostasis in neurons. Beyond the study of progranulin deficiency, these newly developed proteomic methods in neurons and brain tissues provided useful tools and data resources for the field to study the highly dynamic neuronal lysosome biology.

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来源期刊
Molecular Neurodegeneration
Molecular Neurodegeneration 医学-神经科学
CiteScore
23.00
自引率
4.60%
发文量
78
审稿时长
6-12 weeks
期刊介绍: Molecular Neurodegeneration, an open-access, peer-reviewed journal, comprehensively covers neurodegeneration research at the molecular and cellular levels. Neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's, and prion diseases, fall under its purview. These disorders, often linked to advanced aging and characterized by varying degrees of dementia, pose a significant public health concern with the growing aging population. Recent strides in understanding the molecular and cellular mechanisms of these neurodegenerative disorders offer valuable insights into their pathogenesis.
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