title: Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.
abstract: 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.
authors.affiliation: Department of Scientific Research and Teaching, Tangshan People's Hospital, Tangshan, 063001, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Song
authors.affiliation: Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Xiong
authors.affiliation: National Biological Experimental Teaching Demonstration Center, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Che
authors.affiliation: Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Qing-Song
authors.initials: QS
authors.lastname: Xi
authors.affiliation: Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Huang
authors.affiliation: Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Hui-Hua
authors.initials: HH
authors.lastname: Xiong
authors.affiliation: Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. pengzhang@tjh.tjmu.edu.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000008:administration & dosage*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D040901:Proteomics; D013388:Succinimides / Q000008:administration & dosage*
chemical_list: D003432:Cross-Linking Reagents; D020033:Protein Isoforms; D021122:Protein Subunits; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D046988:Proteasome Endopeptidase Complex
keywords: breast cancer; chemical cross-linking; proteasome; protein-protein interaction; threedimensional gel electrophoresis