ORF21-01 - A Golgi-ER Ubiquitin Network Governing PD-L1 Stability in Islet Beta Cells

Type 1 diabetes (T1D) is characterized by the selective destruction of pancreatic beta cells by autoreactive T cells. Expression of the immune checkpoint molecule PD-L1 on beta cells is critical for maintaining self-tolerance by binding to its receptor PD-1 on T cells, thereby inhibiting T cell activity and suppressing autoimmune responses. We previously reported that GOLM1 stabilizes PD-L1 by suppressing its ubiquitin-mediated degradation. However, the specific mechanism by which GOLM1 stabilizes PD-L1 remains unclear. In this study, we leveraged proteomics, RNA sequencing, small interfering RNAs (siRNAs), and immunoblot analysis using immortalized human beta cells (EndoC-betaH1) and human islets to test the hypothesis that a GOLM1-ubiquitin-proteasome-dependent mechanism posttranslationally governs PD-L1 levels. Proteomics analysis revealed that levels of GOLM1 and the E3 ubiquitin ligase TRIM21 are increased in EndoC-betaH1 cells and human islets following exposure to pro-inflammatory cytokines (IFN-gamma and IL-1beta), a treatment paradigm that mimics the inflammatory milieu of T1D. Given their established roles in ubiquitin-dependent protein turnover, we tested whether GOLM1 and TRIM21 function as post-translational regulators of PD-L1 abundance in beta cells. Immunoprecipitation studies demonstrated that TRIM21 physically associates with PD-L1, and siRNA-mediated knockdown of TRIM21 revealed that TRIM21 promotes PD-L1 ubiquitination and proteasomal degradation. Consistent with our prior observations, GOLM1 stabilizes PD-L1 by limiting its ubiquitin-proteasome–mediated turnover. We next identified POFUT1, an ER-resident fucosyltransferase, as a critical intermediary in this pathway. POFUT1 simultaneously interacts with PD-L1 and TRIM21, forming a ternary complex that limits TRIM21-dependent ubiquitination of PD-L1 and thereby preserves PD-L1 protein abundance. Disruption of POFUT1 enhanced TRIM21-mediated PD-L1 degradation, whereas GOLM1 positively regulated POFUT1 expression, positioning GOLM1 upstream in this regulatory cascade. Collectively, these findings establish a GOLM1–POFUT1–TRIM21 network that post-translationally stabilizes PD-L1 in beta cells by restraining ubiquitin-mediated proteasomal degradation. This mechanism provides new insight into how beta cells preserve immune checkpoint signaling in inflammatory settings relevant to T1D.