Walker Jada N, Gautam Amit K S, Matouschek Andreas, Brodbelt Jennifer S
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States.
J Proteome Res. 2024 Dec 6;23(12):5438-5448. doi: 10.1021/acs.jproteome.4c00568. Epub 2024 Oct 30.
Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases. Devising methods to characterize the structures of 20S proteasomes provides necessary insight for the development of drugs and inhibitors that restore proper proteasomal function. Here, native mass spectrometry was combined with multiple dissociation techniques, including ultraviolet photodissociation (UVPD), to identify the protein subunits comprising the 20S proteasome. UVPD, demonstrating an ability to uncover structural features of large (>300 kDa) macromolecular complexes, provided complementary information to conventional collision-based methods. Additionally, variable-temperature electrospray ionization was combined with UV photoactivation to study the influence of solution temperature on the stability of the 20S proteasome.
由于20S蛋白酶体在多种病理过程中发挥作用，包括神经退行性疾病、蛋白酶体相关自身炎症综合征（PRAAS）和心血管疾病，了解其结构和组成如何导致功能障碍至关重要。作为一种735 kDa的蛋白质组装体，20S蛋白酶体通过降解氧化和错误折叠的蛋白质来促进正常的细胞蛋白质稳态。蛋白酶体活性下降可归因于20S蛋白酶体结构完整性的破坏，被认为是多种蛋白酶体相关疾病的主要促成因素之一。设计表征20S蛋白酶体结构的方法为开发恢复蛋白酶体正常功能的药物和抑制剂提供了必要的见解。在此，将天然质谱与多种解离技术相结合，包括紫外光解离（UVPD），以鉴定构成20S蛋白酶体的蛋白质亚基。UVPD能够揭示大型（>300 kDa）大分子复合物的结构特征，为传统的基于碰撞的方法提供了补充信息。此外，将可变温度电喷雾电离与紫外光活化相结合，以研究溶液温度对20S蛋白酶体稳定性的影响。
