Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

被引:5
|
作者
Rao, Geeta [1 ]
Nkepang, Gregory [1 ]
Xu, Jian [2 ]
Yari, Hooman [1 ]
Houson, Hailey [1 ]
Teng, Chengwen [1 ]
Awasthi, Vibhudutta [1 ]
机构
[1] Univ Oklahoma, Hlth Sci Ctr, Dept Pharmaceut Sci, Oklahoma City, OK 73104 USA
[2] Univ Oklahoma, Hlth Sci Ctr, Dept Med, Oklahoma City, OK USA
来源
FRONTIERS IN CHEMISTRY | 2018年 / 6卷
关键词
proteasome; CLEFMA; EF24; RPN13; proteasome inhibitors; dienone; diphenyldihaloketone; NF-KAPPA-B; MULTIPLE-MYELOMA; 2-DIMENSIONAL ELECTROPHORESIS; PROTEIN INTERACTIONS; SIGNALING PATHWAY; HEMORRHAGIC-SHOCK; CANCER CELLS; AUTOPHAGY; IDENTIFICATION; LOCALIZATION;
D O I
10.3389/fchem.2018.00392
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin- like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin- like proteasome activities.
引用
收藏
页数:13
相关论文
共 33 条
  • [21] Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13
    Ting Du
    Yan Song
    Arghya Ray
    Dharminder Chauhan
    Kenneth C. Anderson
    Leukemia, 2021, 35 : 550 - 561
  • [22] Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome
    Seong, Ki Moon
    Baek, Je-Hyun
    Yu, Myeong-Hee
    Kim, Joon
    FEBS LETTERS, 2007, 581 (13) : 2567 - 2573
  • [23] Proteomic Analysis Identifies Mechanism(s) of Overcoming Bortezomib-Resistance Via Targeting Ubiquitin Receptor Rpn13
    Du, Ting
    Song, Yan
    Ray, Arghya
    Chauhan, Dharminder
    Anderson, Kenneth C.
    BLOOD, 2019, 134
  • [24] Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis (vol 58, pg 1053, 2015)
    Marshall, Richard S.
    Li, Faqiang
    Gemperline, David C.
    Book, Adam J.
    Vierstra, Richard D.
    MOLECULAR CELL, 2021, 81 (09) : 2053 - 2053
  • [25] 1H, 13C and 15N resonance assignments of Rpn9, a regulatory subunit of 26S proteasome from Saccharomyces cerevisiae
    Yujie Wu
    Yunfei Hu
    Changwen Jin
    Biomolecular NMR Assignments, 2014, 8 : 307 - 311
  • [26] 1H, 13C and 15N resonance assignments of Rpn9, a regulatory subunit of 26S proteasome from Saccharomyces cerevisiae
    Wu, Yujie
    Hu, Yunfei
    Jin, Changwen
    BIOMOLECULAR NMR ASSIGNMENTS, 2014, 8 (02) : 307 - 311
  • [27] 1H, 13C and 15N resonance assignments of the VWA domain of Saccharomyces cerevisiae Rpn10, a regulatory subunit of 26S proteasome
    Wu, Yujie
    Hu, Yunfei
    Jin, Changwen
    BIOMOLECULAR NMR ASSIGNMENTS, 2014, 8 (02) : 391 - 394
  • [28] 1H, 13C and 15N resonance assignments of the VWA domain of Saccharomyces cerevisiae Rpn10, a regulatory subunit of 26S proteasome
    Yujie Wu
    Yunfei Hu
    Changwen Jin
    Biomolecular NMR Assignments, 2014, 8 : 391 - 394
  • [29] Interaction of hHR23 with S5a - The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome
    Hiyama, H
    Yokoi, M
    Masutani, C
    Sugasawa, K
    Maekawa, T
    Tanaka, K
    Hoeijmakers, JHJ
    Hanaoka, F
    JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (39) : 28019 - 28025
  • [30] Ubiquitin-Like protein 5 interacts with the silencing suppressor p3 of rice stripe virus and mediates its degradation through the 26S proteasome pathway
    Chen, Binghua
    Lin, Lin
    Lu, Yuwen
    Peng, Jiejun
    Zheng, Hongying
    Yang, Qiankun
    Rao, Shaofei
    Wu, Guanwei
    Li, Junmin
    Chen, Zhuo
    Song, Baoan
    Chen, Jianping
    Yan, Fei
    PLOS PATHOGENS, 2020, 16 (08)