ROLE OF ER CHAPERONE BIP/GRP78 & MODIFIED FORMS IN ?-CELL DEATH, TYPE 1 DIABETES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 1型糖尿病是一种自身免疫性疾病，分泌胰岛素的细胞被渗透的免疫细胞破坏。Mathieu实验室最近的研究揭示了参与这种免疫介导的细胞死亡的重要信号通路。这些研究是在炎性细胞因子白介素1和干扰素-伽马治疗后，对产生胰岛素的INS-1E细胞进行蛋白质组2D-DGE分析(D‘Hertog等人，2007年)。对差异表达蛋白质的相互作用组网络分析显示，一些有趣的蛋白质位于该网络的中心位置，提示在诱导细胞死亡方面起着关键作用。其中的关键蛋白之一是葡萄糖调节蛋白78(GRP78/Bip)，它是一种在细胞中高度表达的内质网伴侣蛋白。虽然这种蛋白的总水平在表达上没有改变(正如Western blotting所分析的那样)，但伴侣蛋白正在经历广泛的翻译后修饰，在细胞因子处理后转向更酸性的形式。与这一发现一致的是，GRP78/Bip至少在葡萄糖饥饿的条件下能够发生邻苯二甲酸化或核糖化。虽然有许多研究表明内质网伴侣蛋白在细胞死亡中起着重要作用，但关于其翻译后修饰状态对功能的影响的信息很少。 本研究的目的是确定GRP78/Bip的PTMS在细胞死亡中的确切功能作用。最近利用ProQ-Diamond以及碱性磷酸酶处理的研究表明，目前的修饰不是由磷酸化引起的。为了进一步调查BiP的PTMS的确切性质和位置，通过广泛的LC-MS/MS表征和生物信息学分析，在CBMS询问了样品。初步结果表明，磷酸化以及核糖化的初步归属。确定GRP78/Bip的PTMS的性质将使Mathieu实验室能够进一步调查这些PTMS的功能影响，例如通过使用特定的药物抑制剂或抑制负责修饰的特定酶的siRNA。此外，这将使这些研究人员能够构建具有这种PTM特定突变的转基因小鼠，通过评估这些小鼠对糖尿病的易感性、细胞因子诱导的体外β细胞死亡(Hoechst/PI染色)、胰岛形态、β细胞胰岛素含量(免疫组织化学)和胰岛素分泌，进一步研究BIP-PTM在体内的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Type 1 diabetes is an auto-immune disease in which the insulin-producing ¿-cells are destroyed by infiltrating immune cells. Recent research from the Mathieu laboratory has revealed important signalling pathways involved in this immune-mediated ¿-cell death. These studies were performed by proteomic 2D-DIGE analysis on insulin-producing INS-1E cells, upon treatment with the inflammatory cytokines interleukin-1 and interferon-gamma (D'Hertog et al, 2007). Interactome network analysis of the differentially expressed proteins, revealed some interesting proteins as very centrally located in this network, suggestive for a crucial role in the induction of ¿-cell death. One of this key proteins is glucose-regulated-protein 78 (or GRP78/BiP), an endoplasmic reticulum chaperone which is highly expressed in ¿-cells. Although total levels of this protein are not altered in expression (as analysed by Western blotting), the chaperone is undergoing extensive post-translational modification, shifting towards a more acidic forms upon cytokine treatment. Consistent with this finding GRP78/BiP has been reported to be able to undergo phorolylation or ribosylation, at least under conditions of glucose starvation. Although there are many studies implicating an important role of ER chaperones in ¿-cell death, very little information is available on the functional impact of their post-translational modification status. The aim of the present study is to determine the precise functional role of the putative PTMs of GRP78/BiP on ¿-cell death. Recent investigations making use of proQ-Diamond, as well as alkaline-phosphatase treatment, suggest that the present modification is not caused by phosphorylation. To further investigate the precise nature and sites of the PTMs for BiP, samples were interrogated at the CBMS via extensive LC-MS/MS characterization and bioinformatics analyses. Preliminary results indicate phosphorylation as well as tentative assignments to ribosylation. Identification of the nature of the PTMs of GRP78/BiP will allow the Mathieu laboratory to further investigate the functional impact of these PTMs, for instance by use of specific pharmacological inhibitors or siRNA silencing of specific enzymes responsible for the modifications. In addition, it will allow these investigators to construct transgenic mice with specific mutation of this PTM, for further investigation of the in vivo role of BIP-PTM by evaluating the susceptibility of these mice to diabetes, cytokine-induced beta-cell death in vitro (Hoechst/PI staining), islet morphology, beta-cell insulin content (immunohistochemistry) and insulin secretion.