Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer

International Journal of Molecular Sciences

Tomo Yonezawa¹  Masamitsu Harada¹  Jun Nagai¹  Takayuki Isagawa²  Koji Maemura³  Hiroaki Semba⁴  Yasuhiro Yoshida⁵  Norihiko Takeda⁶  Riho Kurata⁷  Xiaofeng Cui⁸ 

[1] Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan;Data Science Center, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke City, Tochigi 329-0498, Japan;Department of Cardiovascular Medicine, Graduate School of Biomedical Sciences, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan;Department of Cardiovascular Medicine, The Cardiovascular Institute, Nishiazabu 3-2-19, Minato-ku, Tokyo 106-0031, Japan;Department of Immunology and Parasitology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan;Division of Cardiology and Metabolism, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke City, Tochigi 329-0498, Japan;Education and Research Center for Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan;School of Chemistry, Chemical Engineering and Life Sciences, School of Materials and Engineering, Wuhan University of Technology, 122 Loushi Rd, Wuhan 430070, China;

关键词: REST;    Sin3B;    PAH1;    SPR;    TR-FRET;   

DOI  :  10.3390/ijms22052323

来源: DOAJ

【 摘 要 】

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

【 授权许可】

Unknown