Sileno Sara, D'Oria Valentina, Stucchi Riccardo, Alessio Massimo, Petrini Stefania, Bonetto Valentina, Maechler Pierre, Bertuzzi Federico, Grasso Valeria, Paolella Katia, Barbetti Fabrizio, Massa Ornella
Research Laboratories, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
Confocal Microscopy Core Facility, Research Laboratory, Bambino Gesù Children's Hospital, IRCSS, Rome, Italy.
J Proteomics. 2014 Jan 16;96(100):314-27. doi: 10.1016/j.jprot.2013.11.011. Epub 2013 Nov 27.
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously we reported that the role of TG2 in insulin secretion may involve cytoplasmic actin remodeling and a regulative action on other proteins during granule movement. The aim of this study was to gain a better insight into the role of TG2 transamidating activity in mitochondria and in the nucleus of INS-1E rat insulinoma cell line (INS-1E) during insulin secretion. To this end we labeled INS-1E with an artificial donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8min. Biotinylated proteins of the nuclear/mitochondrial-enriched fraction were analyzed using two-dimensional electrophoresis and mass spectrometry. Many mitochondrial proteins involved in Ca(2+) homeostasis (e.g. voltage-dependent anion-selective channel protein, prohibitin and different ATP synthase subunits) and many nuclear proteins involved in gene regulation (e.g. histone H3, barrier to autointegration factor and various heterogeneous nuclear ribonucleoprotein) were identified among a number of transamidating substrates of TG2 in INS-1E. The combined results provide evidence that a temporal link exists between glucose-stimulation, first phase insulin secretion and the action of TG on histone H3 both in INS-1E and human pancreatic islets.
Research into the role of transglutaminase 2 during insulin secretion in INS-1E rat insulinoma cellular model is depicting a complex role for this enzyme. Transglutaminase 2 acts in the different INS-1E compartments in the same way: catalyzing a post-translational modification event of its substrates. In this work we identify some mitochondrial and nuclear substrates of INS-1E during first phase insulin secretion. The finding that TG2 interacts with nuclear proteins that include BAF and histone H3 immediately after (2-5min) glucose stimulus of INS-1E suggests that TG2 may be involved not only in insulin secretion, as suggested by our previous studies in cytoplasmic INS-1E fraction, but also in the regulation of glucose-induced gene transcription.
转谷氨酰胺酶2（TG2）是一种具有钙依赖性转酰胺基和G蛋白活性的多功能蛋白质。此前我们报道，TG2在胰岛素分泌中的作用可能涉及细胞质肌动蛋白重塑以及在颗粒移动过程中对其他蛋白质的调节作用。本研究的目的是更深入了解TG2转酰胺基活性在胰岛素分泌过程中对INS-1E大鼠胰岛素瘤细胞系（INS-1E）线粒体和细胞核的作用。为此，我们在基础条件下以及用葡萄糖刺激2、5和8分钟后，用一种人工供体（生物素化肽）标记INS-1E。使用二维电泳和质谱分析富含核/线粒体部分的生物素化蛋白质。在INS-1E中，在TG2的许多转酰胺基底物中鉴定出了许多参与钙稳态的线粒体蛋白（如电压依赖性阴离子选择性通道蛋白、抑制素和不同的ATP合酶亚基）以及许多参与基因调控的核蛋白（如组蛋白H3、自身整合障碍因子和各种不均一核核糖核蛋白）。综合结果提供了证据，表明在INS-1E和人胰岛中，葡萄糖刺激、第一相胰岛素分泌以及TG对组蛋白H3的作用之间存在时间上的联系。
对转谷氨酰胺酶2在INS-1E大鼠胰岛素瘤细胞模型胰岛素分泌过程中作用的研究描绘了这种酶的复杂作用。转谷氨酰胺酶2以相同方式在INS-1E的不同区室中起作用：催化其底物的翻译后修饰事件。在这项工作中，我们鉴定了第一相胰岛素分泌过程中INS-1E的一些线粒体和核底物。在INS-1E受到葡萄糖刺激后立即（2 - 5分钟），TG2与包括BAF和组蛋白H3在内的核蛋白相互作用，这一发现表明TG2可能不仅如我们之前在细胞质INS-1E部分的研究所表明的那样参与胰岛素分泌，还参与葡萄糖诱导的基因转录调控。
