Chidawanyika Tamutenda, Sergison Elizabeth, Cole Michael, Mark Kenneth, Supattapone Surachai
1Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 USA.
2Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03756 USA.
Cell Death Discov. 2018 Dec 18;4:115. doi: 10.1038/s41420-018-0135-5. eCollection 2018.
Endoplasmic reticulum (ER) stress from accumulated misfolded proteins in the ER can activate the unfolded protein response (UPR). The UPR acts either to restore proteostasis or to activate cell death pathways if the stress cannot be resolved. The key downstream effectors in these pathways have been studied extensively. However, in comparison, stressor-specific key mediators are not as well characterized. In this study, we sought to identify and compare the genes that are necessary for cell death induced by three classic pharmacological ER stressors with different mechanisms of action: thapsigargin, tunicamycin, and brefeldin A. We conducted genome-wide CRISPR/Cas9-based loss-of-function screens against these agents in HAP1 cells, which are a near-haploid cell line. Our screens confirmed that MFSD2A and ARF4, which were identified in previous screens, are necessary for tunicamycin- and brefeldin A-induced cytotoxicity, respectively. We identified a novel gene, SEC24A, as an essential gene for thapsigargin-induced cytotoxicity in HAP1 cells. Further experiments showed that the ability of SEC24A to facilitate ER stress-induced cell death is specific to thapsigargin and that SEC24A acts upstream of the UPR. These findings show that the genes required for ER stress-induced cell death are specific to the agent used to induce ER stress and that the resident ER cargo receptor protein SEC24A is an essential mediator of thapsigargin-induced UPR and cell death.
内质网（ER）中积累的错误折叠蛋白所引发的内质网应激能够激活未折叠蛋白反应（UPR）。如果应激无法得到解决，UPR要么恢复蛋白质稳态，要么激活细胞死亡途径。这些途径中的关键下游效应器已得到广泛研究。然而，相比之下，应激源特异性的关键介质尚未得到很好的表征。在本研究中，我们试图鉴定并比较由三种具有不同作用机制的经典药理学内质网应激源（毒胡萝卜素、衣霉素和布雷菲德菌素A）诱导细胞死亡所必需的基因。我们在近乎单倍体细胞系HAP1细胞中针对这些药物进行了基于全基因组CRISPR/Cas9的功能丧失筛选。我们的筛选证实，先前筛选中鉴定出的MFSD2A和ARF4分别是衣霉素和布雷菲德菌素A诱导细胞毒性所必需的。我们鉴定出一个新基因SEC24A，它是HAP1细胞中毒胡萝卜素诱导细胞毒性的必需基因。进一步的实验表明，SEC24A促进内质网应激诱导细胞死亡的能力是毒胡萝卜素特有的，并且SEC24A在UPR上游起作用。这些发现表明，内质网应激诱导细胞死亡所需的基因因用于诱导内质网应激的药物而异，并且驻留在内质网的货物受体蛋白SEC24A是毒胡萝卜素诱导的UPR和细胞死亡的关键介质。
