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Functional studies of the sle-risk genes bank1 and blk in b-cell pathways

  • Autores: Alejandro Diaz Barreiro
  • Directores de la Tesis: Marta Alarcón (dir. tes.)
  • Lectura: En la Universidad de Granada ( España ) en 2017
  • Idioma: español
  • Tribunal Calificador de la Tesis: Jaime Sancho López (presid.), Mª Carmen Ruiz Ruiz (secret.), Ana Clara Carrera Ramírez (voc.), Juan Sainz Pérez (voc.), Daniel Krappmann (voc.)
  • Materias:
  • Resumen
    • Systemic lupus erythematosus (SLE) is a multisystemic and chronic disorder considered as the prototype autoimmune disease. It is characterized by the production of autoantibodies against nuclear self-antigens. Subsequent deposition of immune complexes in end-organs such as kidneys, joints or skin causes inflammation and tissue damage. The factors that lead to the onset of SLE are several and of different nature. The disease develops when environmental factors, such as infections or ultraviolet light, affect individuals genetically predisposed to encourage the breakdown of tolerance to ubiquitous self-antigens. In this sense, the study of the genetic basis of lupus has been of enormous importance to describe the processes that underlie its pathophysiology.

      During the last decade, the development of genome wide association studies (GWAS) and the concomitant technological advances have led to the identification of more than 60 loci associated with the risk of developing lupus. Most of them are genes encoding proteins with a role in the innate and/or the adaptive immune responses, and as a whole, this information displays which functional pathways are altered in the disease. However, much of this knowledge will not have a medical application unless we accurately figure out in what molecular networks and how these proteins contribute to the development of autoimmunity. Biological therapies targeting B lymphocytes for their modulation or depletion are a clear example of this. Their development would not have been possible without prior basic research such as the one presented here.

      B lymphocyte kinase (BLK) and the B-cell scaffold protein with ankyrin repeats BANK1 are two examples of genes consistently associated with SLE through GWAS. In addition, both proteins show an expression almost restricted to B lymphocytes and have been shown to act together upon B cell receptor (BCR) signaling.

      In 2012 our group identified a rare variant associated with lupus in the BLK gene. A substitution of an alanine for a threonine at position 71 (A71T), within the SH3 domain of the protein, generated a kinase with increased susceptibility to degradation. In order to describe in depth the effects of this non-synonymous variant over BLK homeostasis and function, we have compared mRNA expression, protein degradation, ubiquitination, subcellular localization and traffic toghetehr with coinmunoprecipitation experiments. The variant increases the in vitro ubiquitination of the kinase and degradation rate in B cells. In addition, it enhances the overall threonine- and tyrosine-phosphorylation of the protein. We hypothesize this is caused by the disruption of an intramolecular interaction between the SH3 domain and the linker segment that joins the kinase and SH2 domains of BLK, which has been shown to regulate the activity of this type of kinases. In addition, it has been described that BLK is ubiquitinated after its activation as a signal for degradation, which supports our hypothesis. On the other hand, the A71T substitution also affects the interaction of BLK with BANK1 by significantly reducing it. In contrast, neither an effect on localization nor subcellular trafficking were observed.

      Low levels of BLK have been repeatedly proposed as a risk factor in the development of SLE. The effect of variant A71T, in this sense, would represent an additive risk effect.

      BANK1 contains a putative TIR domain sequence. Our group has intensively investigated its involvement in the Toll-like receptor pathway. We have previously reported BANK1 effects as a positive regulator of TLR7 and TLR9 signaling in mouse models, however little is known about its exact functions. Two isoforms of BANK1 have been described: the Full Length (FL) and the so-called Delta 2 (D2), which lacks the TIR domain-encoding exon 2. TIR domains are key players in TLRs signaling pathways, being found for example in the master regulator MyD88. In addition, we have also identified several TRAF6 putative binding sites within BANK1 sequence. In order to test the functionality of all these domains, we performed coinmunoprecipitation assays with both proteins, MyD88 and TRAF6. The D2 variant displayed a decreased ability to bind both proteins, suggesting that the TIR domain is functional, and that some TRAF6 binding site/s may be affected by alternative splicing of the isoform. On the other hand, we found that FL BANK1 enhances TRAF6 Lys-63-linked ubiquitination, whereas D2 fails to induce it probably because of the altered binding to MyD88 and TRAF6.

      Taken together, these data implicate BANK1 in the TLR pathway and suggest its role as a signaling enhancer mediated through the interaction with MyD88 and TRAF6, particularly by promoting TRAF6 Lys-63-linked ubiquitination.

      The description of pathological pathways such as those studied in this work together with other findings achieved daily in the world's laboratories will allow the development of new personalized and effective therapies for SLE.

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