GRASP55 Regulates Pancreatic Beta-cell Golgi Structure and Function During Cytokine Stress

ABSTRACT. Type 1 Diabetes (T1D) results from autoimmune-mediated destruction of pancreatic β-cells. Currently, no long- term treatments have been successful in preventing disease, and β-cell contribution to early pathology is poorly understood. Early defects in β-cell secretory function, such as mis-trafficking of secretory proteins, are evident prior to symptomatic onset. These defects are suggested to play a role in early disease progression, possibly by neoantigen formation, yet underlying mechanisms are poorly understood. Our recent work demonstrates that proinflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), substantially disrupt β-cell Golgi structure and function, which may explain early defects in β-cell secretory function. The purpose of this proposal is to investigate how cytokine exposure drives alterations to Golgi structure, and the implications this has on Golgi functions. To this point, we demonstrate treatment with a chemical NO donor can recapitulate altered β-cell Golgi structure and function. Our preliminary data further demonstrates knockdown (KD) of GRASP55, a key Golgi structural protein, can substantially block Golgi fragmentation during cytokine stress, which may be regulated via post-translational modification. Our central hypothesis is that proinflammatory cytokines drive NO-dependent, GRASP55- mediated Golgi remodeling, leading to dysfunctional protein sorting and glycosylation. We will test this hypothesis through two aims. Aim 1) will investigate the role of iNOS and NO in altered Golgi structure and cell-surface glycosylation through genetic and pharmacological models of iNOS inhibition, as well as flow cytometry to measure cell surface glycosylation. Aim 2) will investigate PTM regulation of GRASP55 in modulation of Golgi structure and improper secretion of CtsD through GRASP55 KD models and expression of PTM-mutants in a GRASP55 knockout (KO) cell line. Pharmacological inhibition of Golgi export will also be used to investigate how GRASP55-dependent Golgi remodeling drives improper CtsD secretion. This proposed work will advance understanding of how proinflammatory cytokines affect β-cell Golgi structure and function, providing novel insight to β-cell contributions to disease pathology early in T1D and potential targets for new therapeutics.