XprG is a putative transcription factor that belongs to the protein group showing the Ndt80-like DNA binding domain and belongs to the p53-like transcription factors family. The p53 transcription factor plays a fundamental role in the regulation of Bcl-2 family of proteins, which in turn regulate mitochondrial events during apoptosis. Preliminary work showed that the XprG transcription factor is involved in cell death induced by paraquat. Therefore, it is of fundamental importance to study the genes that are activated by XprG transcription factor to better understand the apoptotic pathway in Aspergillus nidulans. Thus, XprG will be characterized, through the study of mutants, regarding its involvement in cell death, evaluating the accumulation of intracellular ROS, mitochondrial function and the sensitivity/resistance to other oxidative stress-inducing substances. Finally, the xrpG mutants and wild type transcriptomes will be compared, both exposed to different times of incubation and concentrations of paraquat and/or conditions of starvation. This experiment aims to identify possible targets, regulated by XprG with subsequent data validation by real time RT-PCR. Afterwards, two or three candidate-genes identified in the screening of microarrays will be functionally characterized by generating and assessing the phenotype of over-expressing mutants, and subcellular localization of proteins encoded by these genes through the generation of fusion proteins with reporter genes. On the other hand, there is evidence that the atypical hexokinases HxkC and HxkD are involved in programmed cell death by regulating the transcription factor XprG. To investigate this hypothesis, double mutants of XprG with HxkC or HxkD fused to different fluorescent proteins will be generated. Using this strategy, it will be possible to visualize in vivo the interaction of the proteins, before and after induction of apoptosis in A. nidulans. Aditionally, the functional characterization of HxkC and HxkD will be performed, through the analyses of mutants, regarding its involvement in cell death, evaluating the accumulation of intracellular ROS, mitochondrial function and the sensitivity/resistance to other oxidative stress-inducing substances.
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