Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue.
Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue.
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DOI：
10.1016/j.jbc.2023.104811
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发表时间：
2023-06
影响因子：
4.8
通讯作者：
Margolis, Seth S
中科院分区：
文献类型：
--
作者：
Turker, Fulya;Bharadwaj, Rahul A;Kleinman, Joel E;Weinberger, Daniel R;Hyde, Thomas M;White, Cory J;Williams, Dionna W;Margolis, Seth S
蛋白酶体是具有多种不同催化活性的大分子复合物，每种催化活性对人类大脑健康和疾病至关重要。尽管它们的重要性，标准化的方法来研究蛋白酶体还没有普遍适应。在这里，我们描述的陷阱，并定义简单的正交生化方法必不可少的测量和了解哺乳动物中枢神经系统中蛋白酶体的组成和活性的变化。通过我们在哺乳动物大脑中的实验，我们确定了大量具有催化活性的蛋白酶体存在，有或没有19S帽，这是泛素依赖性降解所必需的调节颗粒。此外，我们了解到，使用基于活性的探针（ABP）的细胞内测量在确定没有19S帽的20S蛋白酶体的可用活性和测量所有神经元蛋白酶体内每个β亚基的单个催化亚基活性方面更灵敏。随后，将这些工具应用于人脑样本，我们惊讶地发现，无论年龄、性别或疾病状态如何，死后组织几乎没有保留19S-加帽的蛋白酶体。在比较阿尔茨海默病（AD）患者和未受影响的个体的脑组织（海马旁回）时，在严重的AD病例中，可用的20 S蛋白酶体活性显著升高，这是以前没有注意到的观察结果。总之，我们的研究建立了标准化的方法，全面调查哺乳动物脑组织中的蛋白酶体，我们揭示了新的见解脑蛋白酶体生物学。
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.