141 Translation and growth pathways are directly influenced by autoimmune regulator (Aire) in skin keratinocytes

Abstract

Autoimmune regulator, Aire, is commonly known to function as a transcriptional regulator in medullary thymic epithelia. However, we and others have identified Aire to express and function in non-thymic tissues, including the skin, where it greatly influences inflammation and tumorigenesis. We report here that, within skin keratinocytes, Aire participates in a variety of cellular processes outside of its classically defined role in the nucleus. We employed a proximity based biotinylation screen (BioID) to examine the localization of Aire and its binding partners within keratinocytes. Aire is observed by structured illumination microscopy to localize in distinct subcellular compartments within the nucleus, cytoplasm, and along cytoskeletal tracks. Disease causing and function blocking mutations in Aire substantially alter these distribution patterns in keratinocytes, suggesting a link between Aire subcellular compartmentalization and pathogenesis. Using an isobaric labeling method (iTRAQ) coupled to tandem mass spectrometry, we identified 295 common Aire binding partners (99.0% probability), most of them novel, and quantitatively assessed the impact of Aire mutations on binding partner associations. Specifically, Aire binding partners associated with protein translation and cell growth (e.g. multiple 40S and 60S ribosomal protein subunits, cell growth-regulating nucleolar protein, eukaryotic translation initiation factors 4 and 5, elongation factor 1 alpha 1) were significantly altered in cells expressing mutant Aire compared to wild-type Aire. Serum pulse assays confirm a positive correlation between loss of wild-type Aire expression in keratinocytes and increased translation associated signaling (P-AKTS473, P-S6KT389, P-S6S235/S236, P-4EBP1S65, P-eIF4BS422, and P-eEF2KS366). These newly identified partners for Aire expand our understanding of Aire function in the skin and may provide a basis for the pro-tumorigenic role for Aire in skin cancers.