A highly sensitive assay for monitoring the secretory pathway and ER stress.
A highly sensitive assay for monitoring the secretory pathway and ER stress.
复制标题
DOI：
10.1371/journal.pone.0000571
复制
发表时间：
2007-06-27
期刊：
影响因子：
3.7
通讯作者：
Tannous BA
中科院分区：
文献类型：
--
作者：
Badr CE;Hewett JW;Breakefield XO;Tannous BA
分泌途径是细胞将蛋白质整合到细胞膜以及将蛋白质分泌到细胞外空间能力的一个关键指标。重要的是，它会因内质网受到应激而被破坏，这种应激可由多种因素诱导，包括突变蛋白质的表达和生理应激。内质网应激反应的激活在许多疾病的病因学中至关重要，例如糖尿病、神经退行性疾病以及癌症。我们开发了一种高度灵敏的检测方法，基于天然分泌的高斯荧光素酶（Gluc）实时监测蛋白质通过分泌途径和内质网应激的加工过程。
将一个Gluc表达盒导入细胞，并通过测量条件培养基中的荧光素酶活性来监测其分泌情况。当这些细胞用在不同步骤干扰分泌途径的药物处理时，Gluc的分泌量可降低多达90%。将Gluc与一种荧光蛋白融合能够实时对分泌途径进行定量和可视化。这种报告蛋白的表达本身不会在细胞中引发内质网应激反应；然而，Gluc在感知这种类型的应激方面非常敏感，这种应激与蛋白质通过分泌途径的加工过程暂时减少有关。与分泌型碱性磷酸酶（SEAP）相比，Gluc分泌检测法的灵敏度高出20000多倍，SEAP是一种用于监测哺乳动物细胞中蛋白质加工和内质网应激的成熟检测方法。
Gluc检测法提供了一种快速、定量且灵敏的技术来监测分泌途径和内质网应激，并且它与高通量筛选的兼容性将有助于发现用于治疗通常会诱导内质网应激的疾病的药物。
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). 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. 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.
登录
查看更多内容
影响因子：
7.8
作者：
Tartakoff, A;Vassalli, P;Detraz, M
通讯作者：
Detraz, M
影响因子：
4.2
作者：
BREAKEFIELD, XO;BRAVERMAN, M;GILLER, EL
通讯作者：
GILLER, EL
影响因子：
56.9
作者：
FORER, A;EMMERSEN, J;BEHNKE, O
通讯作者：
BEHNKE, O
影响因子：
4.8
作者：
Markova, SV;Golz, S;Vysotski, ES
通讯作者：
Vysotski, ES
影响因子：
82.9
作者：
Iwawaki, T;Akai, R;Miura, M
通讯作者：
Miura, M