Protein disulfide bond formation in many organisms ranging from bacteria to humans is mediated by glutathione redox buffers

Protein disulfide bond formation in many organisms ranging from bacteria to humans is mediated by glutathione redox buffers. This process requires a particular redox potential which depends upon the ratios of glutathione reduced and oxidized moieties. A balance between these ratios, and hence redox potential, is important for maintenance of redox homeostasis across organelles. The redox potential of Endoplasmic Reticulum (ER) is oxidizing in nature and hence suitable for proper disulfide bond formations in proteins. Since, there are different cells with different protein folding load in ER, there might be differences in their redox potential as well. However, tissue specific differences and regulation of redox potential of ER is poorly understood. Here, using genetically encoded roGFP (redox-sensitive Green Fluorescent Protein) sensor in Zebrafish (Danio rerio), we want to find out the tissue specific changes in redox potential of ER. It responds to glutathione couple and is targeted to ER in transgenic line. We checked localization of roGFP and found that it is enriched in ER. The roGFP fishes are physiologically normal as stress markers, like GRP78 and GRP94, and ROS levels are not higher as compared to control. We imaged the embryos, calculated the roGFP ratios using MATLAB and found some regions with different roGFP ratios. Additionally, as proteostasis in ER is maintained by Unfolded Protein Response (UPR) pathways, we also aim to find the role of three core UPR sensors, i.e., IRE1, PERK and ATF6 in regulation of redox homeostasis of ER. Morpholino based knockdown of IRE1 has been done and several developmental defects are found in the embryos. Imaging will be done at lower doses to comment further.

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