Song Hai, Xiong Hua, Che Jing, Xi Qing-Song, Huang Liu, Xiong Hui-Hua, Zhang Peng
Department of Scientific Research and Teaching, Tangshan People's Hospital, Tangshan, 063001, China.
Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2016 Aug;36(4):564-570. doi: 10.1007/s11596-016-1626-3. Epub 2016 Jul 28.
The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.
泛素-蛋白酶体系统通过控制转录因子在乳腺肿瘤发生中起关键作用，从而促进细胞周期生长以及肿瘤抑制蛋白的降解。然而，乳腺癌患者未能从蛋白酶体抑制剂治疗中获益，部分原因是蛋白酶体的异质性，而在恶性乳腺肿瘤中对其了解甚少。化学交联是绘制蛋白质三维结构和蛋白质-蛋白质相互作用图谱的一种日益重要的工具。在本研究中，使用两种交联剂，双（磺基琥珀酰亚胺）辛二酸酯（BS(3)）及其水不溶性类似物二琥珀酰亚胺辛二酸酯（DSS）来绘制MDA-MB-231细胞20S蛋白酶体核心颗粒（CP）中的亚基-亚基相互作用图谱。使用了不同类型的凝胶电泳技术。结合化学交联和质谱，我们将这些凝胶电泳技术应用于研究20S蛋白酶体亚基之间的非共价相互作用。首先，对CP亚基异构体进行了分析。随后，使用天然/SDS-PAGE观察到，0.5 mmol/L BS(3)是用于CP亚基-亚基相互作用研究的相对最佳交联浓度。对交联后的CP进行二维电泳分析表明，α1可能与α2预先相互作用，α3可能与α4预先相互作用。此外，由于蛋白酶体的异质性，α1α2和α3α4存在不同的亚型。BS(3)和DSS之间CP亚基的交联模式没有显著差异。综上所述，基于凝胶的表征与化学交联相结合可作为研究多亚基蛋白质复合物内亚基相互作用的工具。在乳腺癌细胞中观察到的20S蛋白酶体亚基的异质性可能为蛋白酶体抑制策略提供一些关键信息。
