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首页> 外文期刊>Molecular & cellular proteomics: MCP >Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross- linking Mass Spectrometry (QXL-MS)
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Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross- linking Mass Spectrometry (QXL-MS)

机译：使用定量交联质谱（QXL-MS）探测人26s蛋白酶体的H2O2介导的人体动力学

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Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of agerelated neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions. Molecular & Cellular Proteomics 18: 954-967, 2019. DOI: 10.1074/ mcp. TIR119.001323.
机译：细胞毒性蛋白质聚集诱导的细胞功能和稳态的损伤是剧中神经变性病理学的标志。由于蛋白酶体降解代表氧化受损蛋白的主要间隙途径，详细了解其应力反应的分子事件对于发展细胞活力和功能的策略至关重要。尽管在氧化应激期间已经显示26s的蛋白酶以拆卸，但其构象动态仍不清楚。为此，我们开发了一种新的数量交联质谱（QXL-MS）工作流程，以响应于氧化应激探讨蛋白酶体复合物的结构动态。该策略包括基于氧化氧化硅代谢标记，基于HB标签的亲和纯化，由体内甲醛和珠子DSSO交联和多级串联质谱（MSN）组成的2步交联反应。识别和量化交联。该综合工作流程已成功应用于探索氧化应激依赖性蛋白酶体调节的分子事件，通过从处理和未处理细胞的蛋白酶体复杂拓扑的比较分析。我们的研究结果表明，H2O2治疗削弱了26s蛋白酶体内的19S-20S相互作用，以及19S和20S子谱系内的重组。完全，这项工作揭示了26s的机械反应，以急性氧化应激，建议在H 2 O 2介导的26s中解离之前的中间蛋白酶体态。这里呈现的QXL-MS策略可以应用于在不同生理条件下研究其他蛋白质复合物的构象变化。分子和细胞蛋白质组学18：954-967,2017。DOI：10.1074 / MCP。 TiR119.001323。

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