La soutenance aura lieu Vendredi 26 janvier à 14h en salle 401 (TBI, bâtiment 39 de l’INSA, 135 Avenue de Rangueil 31400 Toulouse).
Thèse intitulée “The three disordered domains of the Knr4 protein and their role in stress hypersensitivity and protein-protein interactions in the yeast Saccharomyces cerevisiae“.
Composition du jury :
- Carine van Heijenoort (rapporteur)
- Humberto Martin Brieva (rapporteur)
- Laurence Nieto (examinateur)
- Nathalie Sibille (examinateur)
- Christophe Dez (invited)
- Laurent Maveyraud (thesis supervisor)
- Didier Zerbib (thesis supervisor)
Knr4/Smi1 is a fungus-specific protein and its deletion makes Saccharomyces cerevisiae and Candida albicans hypersensitive to specific antifungals and cell wall stresses. Signalling pathways play an essential role in the response to cellular stress. In S. cerevisiae, the Knr4 hub protein is located at the crossroads of several signalling pathways. This thesis focuses on its crucial role in stress response and resistance to antifungal agents. Knr4 interacts genetically and physically with numerous proteins, including proteins of the Cell Wall Integrity (CWI) and Calcineurin pathways. Small-angle X-ray scattering and crystallographic analysis showed that Knr4 is composed of a structured core domain interrupted by a small disordered loop and flanked by large disordered N- and C-termini. Given the results obtained on other intrinsically disordered proteins (IDPs), such as the tumour suppressor p53, it was hypothesised that the disordered domains of Knr4 are functionally significant.
Chromosomal mutants of Knr4 were constructed using the CRISPR/Cas9 technique, in which one or multiple of the disordered domains of Knr4 were deleted. Cell resistance to different types of stress factors was assessed by growth assays in liquid and solid media. Results revealed that the N-terminal domain and loop are essential for optimal resistance to cell wall binding agents Congo red and calcofluor white, the antifungal caspofungin, high temperature or chemicals such as sorbic acid (acid stress), LiCl (ionic stress), ethanol or the purine analogue Caffeine. Deletion of the C-terminus of Knr4 resulted in increased resistance to cell wall-binding stressors (Congo red and calcofluor white), but not to the other chemical and physical stressors tested. In addition to their influence on stress resistance, the necessity of the N-terminus and the negative influence of the C-terminus of Knr4 were demonstrated in the ability of Knr4 to interact with one of its partner proteins Slt2, the MAPK of the CWI pathway. These results led to the hypothesis that the interaction of the disordered domains of Knr4 with partner proteins such as Slt2 is necessary for the function of Knr4 in stress resistance.
NMR titration experiments with another known partner protein, Tys1, have suggested a low binding affinity between Knr4 and Tys1 and indicated some potential interaction regions in the N-terminal domain and loop of Knr4. We suggest that the interaction regions identified could also be involved in the Knr4-Slt2 interaction. It is even possible that these regions in the disordered domains of the Knr4 hub are also involved in interaction with other of its partner proteins.
Upon induction of the CWI pathway, Slt2 is phosphorylated and enters the nucleus to activate gene transcription. We have shown that simultaneous deletion of the N-terminus and loop of Knr4 increases both total Slt2 levels and Slt2 phosphorylation, a result also seen for the knr4∆ strain. This suggests a potential inhibitory role for Knr4 on the presence of phosphorylated Slt2 which is an indicator of CWI pathway activation.
In conclusion, this thesis has identified the disordered domains of the Knr4 hub protein as modulators of protein function in stress resistance to multiple stressors as well as in the interaction with one of its key partner proteins, the Slt2 MAPK. Some of these stress factors cause an increase in Slt2 phosphorylation and hence CWI signalling, a phenotype also found in mutants deleted for the N-terminus and loop of Knr4. This leads us to hypothesise that the Knr4 IDP could act as a potential (direct or indirect) regulator for the downregulation of the CWI pathway with an essential role for the disordered domains of the protein. Based on the hub nature of Knr4, interaction with other partner proteins could be regulated by the same disordered domains, suggesting that these Knr4 domains could be involved in different functions of the protein.