Kunjunni Remesh, Sathianathan Sandeep, Behari Madhuri, Chattopadhyay Parthaprasad, Subbiah Vivekanandhan
Department of Neurobiochemistry, India Institute of Medical Sciences, New Delhi, -110029, India.
Department of Neurology, India Institute of Medical Sciences, New Delhi, -110029, India.
Biol Trace Elem Res. 2016 Jul;172(1):120-126. doi: 10.1007/s12011-015-0577-z. Epub 2015 Dec 10.
Copper is an essential microelement required for maintaining normal cell physiology. Copper transporter CutC is one of the six members of Cut family proteins, involved in prokaryotic copper homeostasis. Human homolog of CutC (hCutC) is an intracellular copper-binding protein with unknown physiological function. In the present study using HepG2 cells, we report the effects of hCutC knockdown on copper sensitivity and morphology of cells that ultimately leads to apoptosis. We silenced hCutC using specific small interfering RNA (siRNA), and its downregulation was confirmed by quantitative real-time PCR. Though there was no significant variation in total cellular copper as estimated by inductively coupled plasma-atomic emission spectrometry (ICP-AES), knockdown of hCutC caused an increase in sensitivity of HepG2 cells to copper loads when compared to control cells (studied by MTT-based cell viability assay). Morphological analysis by transmission electron microscopy (TEM) indicated onset of apoptosis in hCutC-silenced cells which was exacerbated upon copper treatment. Mitochondrial transmembrane potential (ΔΨm) assay and DNA fragmentation assay further ensured apoptosis occurring in cells upon hCutC silencing. The present study reveals copper induced damage in cells upon hCutC silencing and provides evidence for the role of hCutC protein in intracellular copper homeostasis.
铜是维持正常细胞生理功能所需的必需微量元素。铜转运蛋白CutC是Cut家族蛋白的六个成员之一，参与原核生物的铜稳态。CutC的人类同源物（hCutC）是一种细胞内铜结合蛋白，其生理功能尚不清楚。在本研究中，我们使用HepG2细胞，报告了hCutC敲低对细胞铜敏感性和形态的影响，最终导致细胞凋亡。我们使用特异性小干扰RNA（siRNA）使hCutC沉默，并通过定量实时PCR证实其下调。尽管通过电感耦合等离子体原子发射光谱法（ICP-AES）估计细胞总铜含量没有显著变化，但与对照细胞相比（通过基于MTT的细胞活力测定法研究），hCutC的敲低导致HepG2细胞对铜负荷的敏感性增加。通过透射电子显微镜（TEM）进行的形态学分析表明，hCutC沉默细胞中出现凋亡，在铜处理后加剧。线粒体跨膜电位（ΔΨm）测定和DNA片段化测定进一步证实了hCutC沉默后细胞中发生凋亡。本研究揭示了hCutC沉默后铜诱导的细胞损伤，并为hCutC蛋白在细胞内铜稳态中的作用提供了证据。
