Protein complex formation and intranuclear dynamics of NAC1 in cancer cells

• Published in: Archives of biochemistry and biophysics Vol. 606; pp. 10 - 15
• Main Authors: Nakayama, Naomi, Kato, Hiroaki, Sakashita, Gyosuke, Nariai, Yuko, Nakayama, Kentaro, Kyo, Satoru, Urano, Takeshi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2016
• Subjects: Cell Nucleus - metabolism; Chromatin - metabolism; Chromatography, High Pressure Liquid; Diffusion; Fluorescence loss in photobleaching (FLIP); Fluorescence Recovery After Photobleaching; Fluorescence recovery after photobleaching (FRAP); Green Fluorescent Proteins - chemistry; HeLa Cells; Histones - chemistry; Humans; Intranuclear dynamics; Kinetics; Live cell photobleaching analysis; Molecular Weight; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Neoplasms - metabolism; Nucleus accumbens-associated protein 1 (NAC1); Protein Binding; Protein complex formation; Protein Domains; Protein Multimerization; Repressor Proteins - genetics; Repressor Proteins - metabolism; Protein complex formation; Nucleus accumbens-associated protein 1 (NAC1); Fluorescence loss in photobleaching (FLIP); Live cell photobleaching analysis; Intranuclear dynamics; Fluorescence recovery after photobleaching (FRAP)
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2016.07.007

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300–500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. •NAC1 forms a 300–500 kDa protein complex or complexes in cancer cells.•NAC1 shows significantly slower recovery kinetics than that of freely diffusing GFP.•NAC1 in the nucleus is associated with or interacts with nuclear proteins or chromatin.•Dimer formation by NAC1 coincides with significantly slower diffusion in the nucleus.