A sensor of protein O-glycosylation based on sequential processing in the Golgi apparatus.
A sensor of protein O-glycosylation based on sequential processing in the Golgi apparatus.
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DOI：
10.1111/tra.12019
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发表时间：
2013-01
期刊：
影响因子：
--
通讯作者：
Linstedt AD
中科院分区：
文献类型：
--
作者：
Bachert C;Linstedt AD
蛋白质o -糖基化在许多过程中都是重要的，包括在选择新合成的蛋白质中通过o -聚糖掩蔽来调节蛋白质水解加工位点。为了研究o -聚糖介导的屏蔽作用，我们制作了一种荧光生物传感器，其o -糖基化位点位于屏蔽furin裂解位点的位置。当o -糖基化不能发生时，传感器被激活，因为氟蛋白裂解释放一个阻断域，允许染料与氟激活蛋白结合。因此，通过设计，糖基化应该阻止furin激活传感器，只有当它首先发生时，这是传统的高尔基结构观点所预测的。事实上，与最近提出的快速分裂模型相反，传感器在正常条件下是非荧光的，但当高尔基复合体去分裂时，传感器变成荧光的。为了测试该传感器作为筛选工具的效用，将表达该传感器的细胞暴露于已知的o -糖基化延伸抑制剂或已知改变糖基化效率的sirna靶向因子中。这些条件激活了传感器，证实了它在识别与蛋白质o -糖基化相关的新抑制剂和细胞因子方面的潜力。总之，这些发现证实了高尔基体中的顺序加工，为研究o -糖基化对蛋白质水解过程的调节建立了一个新的工具，并证明了该传感器在未来筛选项目中的潜在用途。
Protein O-glycosylation is important in numerous processes including the regulation of proteolytic processing sites by O-glycan masking in select newly synthesized proteins. To investigate O-glycan-mediated masking using an assay amenable to large-scale screens, we generated a fluorescent biosensor with an O-glycosylation site situated to mask a furin cleavage site. The sensor is activated when O-glycosylation fails to occur because furin cleavage releases a blocking domain allowing dye binding to a fluorogen activating protein. Thus, by design, glycosylation should block furin from activating the sensor only if it occurs first, which is predicted by the conventional view of Golgi organization. Indeed, and in contrast to the recently proposed rapid partitioning model, the sensor was non-fluorescent under normal conditions but became fluorescent when the Golgi complex was decompartmentalized. To test the utility of the sensor as a screening tool, cells expressing the sensor were exposed to a known inhibitor of O-glycosylation extension or siRNAs targeting factors known to alter glycosylation efficiency. These conditions activated the sensor substantiating its potential in identifying new inhibitors and cellular factors related to protein O-glycosylation. In sum, these findings confirm sequential processing in the Golgi, establish a new tool for studying the regulation of proteolytic processing by O-glycosylation, and demonstrate the sensor’s potential usefulness for future screening projects.
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影响因子：
46.9
作者：
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