Characterization of SNX27 function in immune synapse formation and activation in T lyphocytes

• Autores: Natalia González Mancha
• Directores de la Tesis: Isabel Mérida San Román (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2021
• Idioma: inglés
• Número de páginas: 211
• Títulos paralelos:
  • Caracterización de la función de SNX27 en la formación de la sinapsis inmune y la activación en linfocitos T
• Tribunal Calificador de la Tesis: Francisco Sánchez Madrid (presid.), Yolanda R. Carrasco (secret.), Claire Michele Hivroz (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Inmunología
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Sorting nexin (SNX) 27 belongs to the SNX family of proteins, which are involved in intracellular trafficking and endosomal signaling. In addition to the conserved phox homology (PX) domain and 4.1/ezrin/radixin/moesin (FERM)-like domains, SNX27 is the only member of the family that contains a unique N-terminal PSD95/Dlg1/ZO-1 (PDZ) domain. This region allows SNX27 to engage proteins bearing a C-terminal class 1 PDZ-binding motif. Besides, it also mediates direct association with the retromer trimer Vps26-Vps35-Vps29. SNX27 association with the retromer and WASH complex facilitates the recycling of PDZ-binding transmembrane cargoes from endosomes to the cell surface, preventing their lysosomal degradation. At the neuronal synapse, SNX27 facilitates the recycling of PDZ-binding receptors, and its defective expression is associated with deficient synaptic function and cognitive impairment. The highly organized structure formed between antigen presenting cells and T lymphocytes is termed immune synapse (IS) by analogy with the neuronal synapse. IS formation upon antigen recognition ensures cell-cell communication and sustained T cell activation. SNX27-enriched organelles in T lymphocytes included recycling endosomes that were found to polarize to the IS, suggesting a mechanism to facilitate the transport of SNX27 cargoes towards this structure.

    In this study we investigated the consequences of SNX27 deficiency in the organization of the T cell IS. We found that SNX27 controls the traffic towards the cell-cell interface of reported PDZ-interactors, including the retromer subunit vacuolar protein sorting protein 26 (Vps26) and diacylglycerol (DAG) kinase (DGK) ζ. SNX27 silencing also abolished the formation of a DAG gradient at the IS, an effect probably related to defects in spatial localization of DGK. Impaired DAG accumulation at the IS did not affect the recruitment of DAG-regulated proteins to this region. However, it affected the correct polarization of the microtubule organizing center (MTOC). SNX27-silenced Jurkat T cells also displayed deficient polarization to the IS of the motor dynactin and the PDZ-interactor centromere protein J (CENPJ), providing additional links to the control of MTOC repositioning. Detailed analysis of cytoskeleton organization showed impaired reorganization of the microtubule network and modest defects in actin clearance at the IS. These alterations as the result of reduced SNX27 expression were accompanied by defects in the arrangement of signaling microclusters at the IS, as well as in the polarization of the secretory machinery towards the T cell stimulator cells (TCS). In contrast to the defects in polarized responses that result from SNX27 deficiency, we found that SNX27 acts as a negative modulator of nuclear factor κB (NF-κB) regulated transcription. This function correlates with the well characterized role of several SNX27 cargoes as negative regulators of NF-κB signaling. Finally, the initial characterization of mice with partial SNX27-deficiency in the T cell compartment revealed normal lymphoid organ development, although we observed a specific increase of the CD8+/CD4+ T cell ratio in spleen and bone marrow. Cytotoxic T lymphocytes from these mice showed enhanced synthesis of lytic enzymes, although this did not result in enhanced effector functions under our ex vivo stimulation conditions. Importantly, MTOC translocation to the IS was found to be hindered in CD4+ T cells, supporting a conservation in this function. Our results broaden the knowledge of SNX27 function in T lymphocytes and strongly support the notion that the SNX27-retromer-WASH complex regulates IS-directed transport of associated cargoes, facilitating IS architecture and correct T cell activation.