Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor

// Yen-Chin Liu 1, * , Chien-Yun Lee 1, 2, 3, * , Chi-Li Lin 4 , Hui-Yi Chen 5, 6 , Guang-Yaw Liu 7, 8 , Hui-Chih Hung 1, 6, 9 1 Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan 2 Graduate Institute of Biotechnology, National Chung-Hsing University (NCHU), Taichung, Taiwan 3 Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan 4 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan 5 Biotechnology Center, National Chung-Hsing University (NCHU), Taichung, Taiwan 6 Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan 7 Institute of Microbiology & Immunology, Chung Shan Medical University, Taichung, Taiwan 8 Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan 9 Institute of Genomics and Bioinformatics, National Chung Hsing University (NCHU), Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Hui-Chih Hung, e-mail: hchung@dragon.nchu.edu.tw Guang-Yaw Liu, e-mail: liugy@csmu.edu.tw Keywords: biochemistry, molecular and cellular biology, signal transduction, cell cycle, oncogene Abbreviations: AZ, antizyme; CCND1, cyclin D1; ODC, ornithine decarboxylase; AZI, antizyme inhibitor; AUC, analytical ultracentrifugation Received: March 06, 2015 Accepted: June 16, 2015 Published: June 26, 2015 ABSTRACT Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant ( K d ) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The K d values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the K d values for CCND1 binding to the AZ N-terminal peptide (AZ 34–124 ) and AZ C-terminal peptide (AZ 100–228 ) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The K d values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.