Clonazione ed espressione eterologa delle protein-chinasi IRAK nel Saccharomyces cerevisiae

The fight against pathogens is one of the most important challenges for an organism and it can be crucial for its survival. The innate immune system in Drosophila and mammals senses the invasion of microorganisms using the family of Toll-like receptors (TLRs). They recognize pathogen-associated molecular patterns and subsequently trigger inflammatory and immune responses. Among the well-conserved components in their signaling pathways are a family of Ser/Thr protein kinases, the Interleukin-1 Receptor-Associated Kinases (IRAKs). In this study these mammalian proteins, were expressed for the first time in Saccharomyces cerevisiae. After a lot of efforts, only three of the four proteins were correctly cloned in the expression vector. The final clones were fully sequenced to verify that no mutations had been introduced during amplification and then transformed in Saccharomyces cerevisiae. First of all, expression was verified by immunoblot and yeast growth assays were performed showing that both IRAK-4 and, at a lower rate, IRAK-1 were able to inhibit yeast cell growth. Upon heterologous IRAK expression, Western blotting revealed phosphorylation of the Slt2 MAPK, a pathway involved in the maintenance of yeast cell wall integrity. Slt2 hyper-activation, however, was not responsible for IRAK-induced toxicity, since IRAK expression in a mutant yeast strain unable to express Slt2 still inhibited growth. To investigate whether IRAK-4 toxicity in yeast was due to its catalytic kinase activity, a site-directed mutagenesis was performed in order to create the kinase-dead (KD) point. Further tests comparing the wild-type and KD mutant revealed that indeed kinase activity was required for growth inhibition and MAPK activation. To understand at which level IRAK 4 activity led to Slt2 activation, we used a series of mutants located upstream Slt2. Finally, several physiological functions, such as actin polarization, endocytic traffic, mitochondrial membrane potential and ROS production were analyzed by cell biological technique such as fluorescence microscopy and cytometry.