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.
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