Song Haowei, Bao Shunzhong, Lei Xiaoyong, Jin Chun, Zhang Sheng, Turk John, Ramanadham Sasanka
Mass Spectrometry Resource, Division of Metabolism, Endocrinology, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biochim Biophys Acta. 2010 May;1801(5):547-58. doi: 10.1016/j.bbalip.2010.01.006. Epub 2010 Feb 2.
Over the past decade, important roles for the 84-88kDa Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) in various organs have been described. We demonstrated that iPLA(2)beta participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA(2)beta, and certain stimuli promote perinuclear localization of iPLA(2)beta. To gain a better understanding of its mobilization, iPLA(2)beta was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA(2)beta by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA(2)beta activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA(2)beta activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA(2)beta isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA(2)beta in beta-cells and we find here that these stimuli promote differential localization of iPLA(2)beta in subcellular organelles. Further, mass spectrometric analyses identified iPLA(2)beta variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA(2)beta and redistribution of iPLA(2)beta variants in subcellular compartments. It might be proposed that in vivo processing of iPLA(2)beta facilitates its participation in multiple biological processes.
在过去十年中，已描述了84 - 88kDa的ⅥA组钙离子非依赖性磷脂酶A2（iPLA2β）在各个器官中的重要作用。我们证明iPLA2β参与胰岛素分泌，胰岛素瘤细胞和天然胰岛表达iPLA2β的全长和截短异构体，并且某些刺激促进iPLA2β的核周定位。为了更好地了解其动员情况，iPLA2β在INS - 1细胞中作为与EGFP的融合蛋白表达，通过监测EGFP荧光能够检测iPLA2β的亚细胞定位。稳定转染融合蛋白的细胞表达的催化性iPLA2β活性比单独转染EGFP cDNA的对照细胞高近5倍，表明EGFP的共表达不干扰iPLA2β活性的表现。对EGFP和细胞器追踪染料的双重荧光监测结合免疫印迹分析揭示了截短的iPLA2β异构体在不同亚细胞器中的表达。已知暴露于促分泌剂和内质网应激诱导会激活β细胞中的iPLA2β，并且我们在此发现这些刺激促进iPLA2β在亚细胞器中的差异定位。此外，质谱分析鉴定了去除N端残基的iPLA2β变体。总的来说，这些发现为iPLA2β的内源性蛋白水解加工以及iPLA2β变体在亚细胞区室中的重新分布提供了证据。可能有人提出，iPLA2β在体内的加工促进了其参与多种生物学过程。
