A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

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A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

dc.contributor.author	Hewett, Jeffrey W.
dc.contributor.author	Badr, Christian
dc.contributor.author	Breakefield, Xandra Owens
dc.contributor.author	Tannous, Bakhos A.
dc.date.accessioned	2011-03-25T15:32:49Z
dc.date.issued	2007
dc.identifier.citation	Badr, Christian E., Jeffrey W. Hewett, Xandra O. Breakefield, and Bakhos A. Tannous. 2007. A highly sensitive assay for monitoring the secretory pathway and ER stress. PLoS ONE 2(6): e571.	en_US
dc.identifier.issn	1932-6203	en_US
dc.identifier.uri	http://nrs.harvard.edu/urn-3:HUL.InstRepos:4773991
dc.description.abstract	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 processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.	en_US
dc.language.iso	en_US	en_US
dc.publisher	Public Library of Science	en_US
dc.relation.isversionof	doi:10.1371/journal.pone.0000571	en_US
dc.relation.hasversion	http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1892804/pdf/	en_US
dash.license	LAA
dc.title	A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress	en_US
dc.type	Journal Article	en_US
dc.description.version	Version of Record	en_US
dc.relation.journal	PLoS ONE	en_US
dash.depositing.author	Badr, Christian
dc.date.available	2011-03-25T15:32:49Z
dash.affiliation.other	HMS^Neurology-Massachusetts General Hospital	en_US
dash.affiliation.other	HMS^Neurology-Massachusetts General Hospital	en_US
dash.affiliation.other	HMS^Neurology-Massachusetts General Hospital	en_US