Browsing by Author "Obidan, Amnah"

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Intragenic suppression of RNase-defective point mutation of the catalytic aspartate in the protein kinase domain of Ire1
(Saudi Digital Library, 2023-12-07) Obidan, Amnah; Schroder, Martin
In eukaryotic cells, proper folding of secretory and transmembrane proteins occurs within the endoplasmic reticulum (ER) before their exit the ER. The accumulation of unfolded proteins activates a response known as the unfolded protein response (UPR), mediated by Ire1. In Saccharomyces cerevisiae, Ire1 activation leads to the splicing of HAC1 mRNA, which encodes a transcription factor involved in the UPR. In this study, we focused on specific mutations within the protein kinase domain of Ire1. Specifically, the protein kinase domain was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the Mg2+ coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. To investigate the impact of these mutations, we performed several experiments. Northern blot analysis was employed to detect the splicing of HAC1 mRNA, as it serves as an indicator of Ire1 activity and UPR induction. Additionally, β-galactosidase reporter assays were conducted to assess the expression of a UPRE-lacZ reporter gene, which is also regulated by the Ire1-Hac1 signalling pathway. The results demonstrated that single mutations in the catalytic domain and Mg2+ coordinating loop (K799A, D797A, N802A, and D828A) led to decreased levels of HAC1 mRNA and reduced expression of the UPRE-lacZ reporter gene compared to the WT Ire1. Furthermore, the D797A mutant strain exhibited decreased survival under ER stress conditions when compared to other mutants within the Mg2+coordinating loop and catalytic domain. Interestingly, the D797A mutation resulted in lower levels of HAC1 mRNA species and β-galactosidase activity. However, introducing additional mutations such as D797A N802A or D797A K799A N802A led to significant increases in βgalactosidase activity, the percentage of HAC1 mRNA, and restored growth compared to the single D797A Ire1 mutant. Notably, the expression levels of WT and protein kinase mutants were similar. In conclusion, the findings suggest that introducing specific additional mutations, such as K799A Ire1, D828A Ire1, or N802A Ire1, to the single D797A Ire1 mutant can restore the signalling activity of Ire1.
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