Transient expression of misfolded surfactant protein C. Homo sapiens

Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. To identify candidate genes involved in ER quality control of SP-C, HEK293 cells were transiently transfected with mutant SP-C (SP-CΔexon4 or SP-CL188Q), SP-CWT, or vector cDNAs, and global changes in gene expression were assessed by microarray analyses. Microarray analysis demonstrated that the SPC exon 4 deletion and SPC L188Q mutations invoke very similar transcriptional profiles including the activation of major players mediating ER response and unfolding protein response (UPR) in transient transfection system. In combination with promoter scan (UPRE, ERSE, XBP1 sites) and protein domain analysis (Finding ER Lumen, ER Membrane retention signal, J-Domain and Leucine Zipper domain), we were able to not only verify the known ERAD components (XBP1, Bip, Erdj4&5), but also identify multiple ER components which may play critical roles in the detection and /or degradation of mutant SPC, which in turn will help us to gain better understanding of the entire mammalian ERAD machinery. Overall design: samples consisted of HEK293 cells transiently transfected with wild-type SP-C (WT), the delta exon4 mutant of SP-C (Ex4) or L188Q mutant of SP-C (LQ). Groups were performed in triplicate.

与人类患者间质性肺疾病相关的SFTPC基因（surfactant protein C gene）突变，会导致其所编码的表面活性蛋白C（surfactant protein C, SP-C）前体蛋白发生错误折叠、内质网（endoplasmic reticulum, ER）滞留及降解。为鉴定参与SP-C内质网质控的候选基因，研究人员将突变型SP-C（SP-CΔexon4或SP-CL188Q）、野生型SP-C（SP-CWT）或空载体的互补DNA（complementary DNA, cDNA）瞬时转染至HEK293细胞中，并通过微阵列（microarray）分析评估基因表达的整体变化。微阵列分析结果显示，SPC exon4缺失突变与SPC L188Q突变所诱导的转录谱极为相似，包括在瞬时转染系统中激活介导内质网应答与未折叠蛋白反应（unfolded protein response, UPR）的核心调控因子。结合启动子扫描（针对UPRE、ERSE、XBP1结合位点）与蛋白质结构域分析（鉴定内质网腔、内质网膜滞留信号、J结构域与亮氨酸拉链结构域），研究人员不仅验证了已知的内质网相关降解（ER-associated degradation, ERAD）组分（XBP1、Bip、Erdj4&5），还鉴定出多种可能在突变型SPC的检测和/或降解中发挥关键作用的内质网组分，这将有助于我们更深入地理解完整的哺乳动物内质网相关降解机制。实验整体设计：实验样本为瞬时转染野生型SP-C（WT）、SP-CΔexon4突变体（Ex4）或SP-CL188Q突变体（LQ）的HEK293细胞，每组设置3次生物学重复。