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Structural anf functional analysis of SpoTAb interactome

par Mansouri, Elisabeth
Promoteur Garcia-Pino, Abel
Co-Promoteur Cepauskas, Albinas
Publication Non publié, 2024-06-24

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Résumé :

This thesis focuses on the study of the AlphaFold predicted interaction of the SpoT protein in Acinetobacter baumannii. SpoT belongs to the long RSH (RelA/SpoT Homologue) family of enzymes, known to be involved in the stringent response. The stringent response is a regulatory mechanism in bacteria that is triggered in response to stressful conditions, such as nutrient deficiency. It is mediated by the (p)ppGpp nucleotide. The long RSH enzymes regulates the levels of this molecules via hydrolysis or synthesis. In this species, SpoT is monofunctional and hydrolyzes (p)ppGpp, facilitating the return to normal conditions.The main aim of this study was to confirm the interactions between SpoT and proteins involved in the translation process and predicted in silico by AlphaFold. To this end, I used the bacterial adenylate cyclase two-hybrid assay, an in vivo interaction test, and confirmed the interaction of 10 proteins out of the 13 initially predicted. In addition, the interaction between SpoT and ACP (Acyl Carrier Protein), already known in the literature to interact in Escherichia coli was also confirmed in Acinetobacter baumannii. Some of these interactions involve GTPases such as EF4 and Era. I, however, focused my study on a specific methyltransferase named RsmC or RNA small subunit methyltransferase C. Characterization of this interaction with SpoT revealed that RsmC enhances SpoT's hydrolase activity. A further characterization of this interaction such as ITC or crystallography studies would be beneficial to understand more deeply this regulation and the overall mechanism within the stringent response in Acinetobacter baumannii.