Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue

被引：9
作者：
Türker F. [1 ]
Bharadwaj R.A. [2 ]
Kleinman J.E. [2 ,3 ]
Weinberger D.R. [2 ,3 ,4 ,5 ]
Hyde T.M. [2 ,3 ,5 ]
White C.J. [6 ]
Williams D.W. [6 ,7 ,8 ,9 ,10 ]
Margolis S.S. [1 ,10 ]
机构：
[1] Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD
[2] The Lieber Institute for Brain Development, Baltimore, MD
[3] Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD
[4] McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD
[5] Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD
[6] Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, Baltimore, MD
[7] Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD
[8] Division of Clinical Pharmacology, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD
[9] Department of Molecular Microbiology & Immunology, Johns Hopkins School of Public Health, Baltimore, MD
[10] Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD
来源：
Journal of Biological Chemistry | 2023年 / 299卷 / 06期
基金：
美国国家卫生研究院;
关键词：
activity-based probe; Alzheimer's disease; mammalian central nervous system; MV151; neurodegeneration; neuron; proteasome; protein degradation; Suc-LLVY-AMC;
D O I：
10.1016/j.jbc.2023.104811
中图分类号：
学科分类号：
摘要：
Proteasomes are large macromolecular complexes with multiple distinct catalytic activities that are each vital to human brain health and disease. Despite their importance, standardized approaches to investigate proteasomes have not been universally adapted. Here, we describe pitfalls and define straightforward orthogonal biochemical approaches essential to measure and understand changes in proteasome composition and activity in the mammalian central nervous system. Through our experimentation in the mammalian brain, we determined an abundance of catalytically active proteasomes exist with and without a 19S cap(s), the regulatory particle essential for ubiquitin-dependent degradation. Moreover, we learned that in-cell measurements using activity-based probes (ABPs) are more sensitive in determining the available activity of the 20S proteasome without the 19S cap and in measuring individual catalytic subunit activities of each β subunit within all neuronal proteasomes. Subsequently, applying these tools to human brain samples, we were surprised to find that post-mortem tissue retained little to no 19S-capped proteasome, regardless of age, sex, or disease state. In comparing brain tissues (parahippocampal gyrus) from patients with Alzheimer's disease (AD) and unaffected individuals, the available 20S proteasome activity was significantly elevated in severe cases of AD, an observation not previously noted. Taken together, our study establishes standardized approaches for the comprehensive investigation of proteasomes in mammalian brain tissue, and we reveal new insight into brain proteasome biology. © 2023 The Authors
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