Análisis proteómico y bioquímico del regulador autoinmune: nuevas funcionalidades y vías de degradación de aire

Abstract

The thymus is the organ where the repertoire of circulating T lymphocytes is generated. An exhaustive selection whitin the thymus is required in order to generate an immunocompetent T cell repertoire: broadly diverse for recognizing pathogens but minimally autoreactive. Central tolerance is based on the successfully maturation of T cells by an appropriate interaction with self Major Histocompatibility Complex (MHC)/peptide complexes. This process consists in a positive selection which takes place in the cortex and a negative selection carried out in the thymic medulla. In the cortex, only thymoctes carrying a functional TCR able to recognize self MHC molecules will survive. In the medulla, thymocytes that recognize self MHC/peptides complexes are eliminated. For negative selection to be efficient it will be necessary the expression of the whole proteome of the body. The Autoimmune Regulator (AIRE) leads the ectopic expression of antigens restricted to tissues and organs (TRAs) by medullary thymic epithelial cells (mTECs). AIRE is a multidomain protein related to the regulation of gene expression and transcription, apoptosis, cell differentiation and cell cycle. Mutations in AIRE gene cause the so-called Autoimmune Polyendocrinopathy type 1 (APS-1), a monogenic and polyorganic autoimmune disease, characterized by the presence of autoantibodies in the blood and lymphocytic infiltrates due to a defective central tolerance process Previous findings of our group showed differences between the proteomes of differentially AIRE expressing cells. AIRE+ cells increased the expression of chaperones and apoptosis-related proteins and dampened cytoskeleton-associated proteins compared to control cells. One of the increased proteins was Calcyclin-Binding Protein or SIAH1-Interacting Protein (CACYBP/SIP), an adaptor protein involved in ubiquitin-proteasome mediated protein degradation. SIP interacts with Seven in Absentia Homologue 1 (SIAH1) E3 ubiquitin ligase through the SIAH1 binding motif. SIAH1 and SIAH2 belongs to SIAH RING-type E3 ubiquitin ligase family. SIAH1 is expressed in response to genotoxic damage while SIAH2 is increased under hypoxia conditions. Both E3 ubiquitin ligases share specific targets, some of them related with AIRE. Protein sequence analysis showed the presence of two possible SIAH1 binding motifs in AIRE sequence: one with the complete sequence and the other with a conservative mutation. In this thesis, some aspects related to AIRE and the effect of its expression in cell lines have been characterized using biochemical and proteomics techniques, which may allow us to find new functions of the protein: First, using quantitative proteomics techniques (iTRAQ) and HLA-I derived peptides repertoire analysis the proteomes and the peptidomes of differentially AIRE expressing cells have been analyzed. A study that had not been carried out up to now. Additionally, the AIRE domains involved in apoptosis have been identify. Finally, this approach has allowed us to show that AIRE induces an increase in cell mobility. Second, a new localization of the AIRE protein has been characterized in post-mitotic cells, surrounding the midbody. Likewise, it has been possible to show the location of AIRE throughout the cell cycle and in different mitotic phases. Third the interaction between AIRE and the E3 ubiquitin ligases of the SIAH family has been biochemically and functionally characterized. AIRE interacts with SIAH1 and SIAH2 through the first interaction motif. SIAH1 leads to the polyubiquitination and degradation of AIRE. That effect is slightly observed in the case of SIAH2 although polyubiquitinated AIRE is also detected.

Date of Award	14 Feb 2023
Original language	Spanish
Supervisor	Iñaki Alvarez (Director)