a highly sensitive assay for monitoring the secretory pathway and er stress一个高度敏感的试验监测分泌通路和er应激.pdfVIP

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress Christian E. Badr1,3., Jeffrey W. Hewett1., Xandra O. Breakefield1,2,3, Bakhos A. Tannous1,2,3* 1 Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, United States of America Background. The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings. An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in pr