| Autor: |
Wires ES; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Henderson MJ; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Yan X; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Bäck S; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Trychta KA; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Lutrey MH; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America., Harvey BK; Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland, United States of America. |
| Abstrakt: |
The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis. |
