Biochimica et Biophysica Acta (BBA) - Bioenergetics ( IF 4.3 ) Pub Date : 2020-08-15 , DOI: 10.1016/j.bbabio.2020.148289 Francesca Guarino 1 , Federica Zinghirino 1 , Lia Mela 1 , Xena Giada Pappalardo 1 , François Ichas 2 , Vito De Pinto 3 , Angela Messina 4
VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.
中文翻译:
NRF-1和HIF-1α有助于在HeLa细胞饥饿和缺氧期间调节人VDAC1基因启动子。
VDAC(电压依赖性阴离子通道)是位于线粒体外膜上的成孔蛋白家族。它的通道特性可确保线粒体与细胞其余部分之间的代谢物交换,从而导致新陈代谢和生物能调节,以及细胞死亡和生命转换。VDAC1是特征最丰富,形式最丰富的同工型,涉及多种病理,如癌症或神经退行性疾病。但是,关于在正常和/或病理条件下其基因表达调控的信息很少。在这项工作中,我们探讨了正常条件下VDAC1基因表达的调控,并以一些代谢和高能的线粒体功能障碍和细胞应激为例。通过生物信息学研究和启动子活性实验,根据转录因子响应元件对推定的启动子区域的核心进行了表征。特别是,我们发现NRF-1位点以及参与细胞生长,增殖,发育的其他转录因子结合位点大量存在,并且我们研究了它们在基因活性中的普遍性。此外,在各种病理学检测到营养耗尽或控制性缺氧后,我们发现VDAC1转录物水平以与时间相关的方式显着增加。VDAC1启动子活性也已通过基因报告基因检测验证。根据PCR实时实验,证实当细胞暴露于应激时,VDAC1启动子活性被进一步刺激。一项生物信息学调查表明,除NRF-1外,HIF-1α还存在 作为最活跃的TFBS。通过TFBS诱变和TF过表达实验获得了它们的验证。总之,我们通过实验证明了NRF-1和HIF-1α参与了基础水平和某些特殊细胞应激条件下VDAC1启动子激活的调节。