SAT068 The Golgi Transmembrane Protein GOLM1 Maintains PD-L1 Protein Levels In Islet Beta Cells In Response To Type 1 Diabetic Inflammation

Abstract

Abstract Disclosure: F. huang: None. J. Nelson: None. C. Muralidharan: None. E. Nakayasu: None. X. Yi: None. D. Eizirik: None. S. May: None. S.A. Tersey: None. R.G. Mirmira: None. In type 1 diabetes (T1D), a dialog between β cells and immune cells results in the dysfunction and autoimmune destruction of β cells. Protein processing pathways in the β cell are critical for the production and processing of immune checkpoint proteins such as PD-L1, which suppress β cell autoimmunity by interacting with receptors (PD-1) on immune cells. We performed unbiased proteomics analysis following proinflammatory cytokine (50 U/ml IL-1β + 1000 U/ml IFN-γ) treatment of the human β cell line EndoC-βH1 to identify potential protein processing molecules. GOLM1, a type II Golgi transmembrane protein involved in the sorting and modification of proteins exported from the endoplasmic reticulum, emerged as a candidate that was increased 33% (p=0.014) in response to cytokines and coordinately upregulated with PD-L1. We hypothesized that GOLM1 is necessary for the posttranscriptional production of PD-L1 in β cells. To test this hypothesis, we performed further quantitative RT-PCR, immunoblot, and flow cytometry studies of GOLM1 and PD-L1 in EndoC-βH1 cells and human islets following cytokine treatment to mimic the inflammatory milieu of T1D. Additional experiments included RNA interference against GOLM1, immunoprecipitation followed by immunoblots, and incubation with an inhibitor of the proteasome (MG132). Treatment of EndoC-βH1 cells with proinflammatory cytokines for 18 h resulted in a 10-fold upregulation of the gene encoding PD-L1. Immunoblot analysis confirmed a >10-fold upregulation of PD-L1 protein, with flow cytometry further indicating that only a subset of β cells (∼60%) exhibit PD-L1 production. GOLM1 protein levels were upregulated nearly 3-fold in response to cytokines in EndoC-βH1 cells and human islets. RNA interference-induced knockdown of GOLM1 in EndoC-βH1 cells abrogated the cytokine-induced increase of PD-L1 protein without altering its corresponding mRNA levels, a finding consistent with a role for GOLM1 in the posttranscriptional regulation of PD-L1 levels. The posttranscriptional effect of GOLM1 on PD-L1 levels appears to involve the proteasome, since proteasome inhibition with MG-132 restored PD-L1 levels in the absence of GOLM1. Direct interaction of GOLM1 with PD-L1 was observed following immunoblot studies in EndoC-βH1 cells. Finally, analysis of the single cell RNA-Seq database of the Human Pancreas Analysis Program revealed a significant increase in GOLM1 in residual β-cells of individuals with T1D compared to controls. Collectively, our data indicate that GOLM1 increases following cytokine-induced inflammation in β cells, stabilizes PD-L1 levels by preventing its proteasomal degradation, and is elevated in residual β cells of individuals with T1D. Our studies provide new insight into the molecular mechanisms by which β cells generate a protective response against autoimmunity in T1D. Presentation: Saturday, June 17, 2023