Effect of zinc chelation in Caco-2 cells

To identify the molecular pathways that are perturbed due to transient zinc chelation Zinc is known to regulate the functions of about 10% of the human proteome and a large number of physiological processes that are zinc dependent have been identified and characterized under conditions of zinc deficiency and supplementation. As zinc homeostasis is closely linked to the normal functioning of both prokaryotic and eukaryotic cells, many pathogens are directly or indirectly affected by perturbations in zinc homeostasis. Dengue virus (DENV), a mosquito-borne, positive-strand RNA virus from the family Flaviviridae, has emerged as one of the major public health concerns in India and recent estimates suggest that over 60 million people globally get infected with DENV every year. The crystal structures of NS5 protein of DENV and West Nile virus have identified zinc binding site in RdRp domain and propose an important structural role for zinc ions in polymerase activity. Therefore, we investigated whether perturbation in intracellular zinc pools influence dengue infection. We utilized N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), a zinc-specific chelator, to mimic zinc-deficiency in cell culture models of infection and investigated the effect of zinc depletion on DENV life-cycle. Overall design: Caco-2 cells were treated with a zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) for four hours and total RNA was isolated and used for RNA sequencing.

为鉴定瞬时锌螯合所扰动的分子通路，本研究开展了相关工作：
已知锌可调控人类蛋白质组约10%的功能，且诸多锌依赖型生理过程已在锌缺乏与锌补充条件下被鉴定并表征。由于锌稳态与原核、真核细胞的正常功能紧密相关，诸多病原体的活性会直接或间接受到锌稳态扰动的影响。
登革病毒（Dengue virus, DENV）是一种经蚊媒传播的黄病毒科正链RNA病毒，现已成为印度主要的公共卫生威胁之一；最新估算显示全球每年有超过6000万人感染DENV。登革病毒与西尼罗河病毒的NS5蛋白晶体结构已在其RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRp）结构域中发现锌结合位点，并提出锌离子在聚合酶活性中发挥关键结构作用。因此，本研究探究了细胞内锌池扰动是否会影响登革病毒感染。
本研究使用锌特异性螯合剂N,N,N',N'-四(2-吡啶甲基)-1,2-乙二胺（N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine, TPEN），在感染细胞培养模型中模拟锌缺乏状态，并探究了锌耗竭对DENV生命周期的影响。
实验整体设计：将Caco-2细胞用锌螯合剂TPEN处理4小时后，提取总RNA并进行RNA测序。