In vivo model uncovers non-canonical UPR mechanisms controlling growth factor biogenesis
Authors:   , Ying Mingjie , Gidalevitz Tali , Sang Nianli 
Published: March 2022

Proteins need to be folded into their native structure to be functional, and this process is called protein folding. Disrupting the folding process leads to misfolded proteins, which will result in loss of function and often cause conformational diseases, such as type II diabetes and neurodegenerative diseases. When the cellular homeostasis is disrupted by the accumulation of misfolded proteins, stress responses are triggered to recover normal steady state. One of them is the unfolded protein response (UPR), the main response that resolves endoplasmic reticulum (ER) stress. Interestingly, the UPR can also can function even without the presence of ER stress. For example, in the biogenesis of insulin, RIDD activity of one of the ER stress sensors, IRE1, is triggered without global activation of the UPR. The general understanding of these UPR functions in the absense of ER stress is still under-studied. The first part of my dissertation is focused on understanding of the folding and secretion of a class of growth factors, insulin/IGF-like proteins. I focused on generating a cell culture-based system for biochemical characterization of the genetic data that our lab generated by using genetic approaches in C. elegans, including the effects of mutations affecting the conserved disulfide bonds on the folding process of insulin/ IGFs family proteins. Second, I developed a biological assay to determine whether the secreted proteins are functional. Thirs, I helped develop imaging approach in cultured neurons to study the effects of ER stress signaling on localization of these growth factors. Even though both the C. elegans DAF-28 and human IGF2 belong to the insulin/IGF family and are predicted to have similar structures, these two proteins are rather different. In this study, both DAF-28 and IGF2 are shown to be secreted from mammalian cells. Interestingly, even though both proteins may bind to the same IIS receptor, secreted DAF-28 may act as an antagonist of IIS signaling, which has opposite function to human IGF2. This idea was indicated by DAF-28 inhibitory effect to the growth of IGF2- dependent cells in the functional assay. Adding an unpaired Cys to IGF2, to mimick the disulfide bonds arrangement of the DAF-28(R37C) mutant, impairs IGF2 folding and secretion. This suggests similar effects of disulfide bonds in the respective protein folds. Interestingly, in neurons, both proteins were trafficked into the same compartment (axon) and were regulated by PERK, an ER stress sensor. Therefore, this study provides evidence that despite difference in the number of Cys residues and low protein sequence similarity, disulfide bonds in members of the insulin/IGF family have similar effects in determining the conformations of the proteins. In the second part of my dissertation, I found that C. elegans utilizes one of UPR activities to protect the survival of its population under environmental stress. In this case, the toxic chemical naturally produced by bacteria encountered by C. elegans is used as a warning signal for the anticipated stress. This protection of survival is achieved by employing regulated IRE1-dependent mRNA decay (RIDD) activity, to selectively downregulate neuronal tgf-b /daf-7 mRNA and thus reduce its signaling. In this the part of dissertation, I showed that C. elegans IRE1 has RIDD capability, by using a chimeric IRE1 that contains C. elegans catalytic domens. Our data demonstrated that C. elegans kinase and RNase domains in the chimeric IRE1 recognized and cleaved the known RIDD substrate Blos-1 mRNA in mammalian cells. This result indicates that both xbp-1 splicing and RIDD are evolutionarily conserved in C. elegans IRE1. After uncovering the C. elegans IRE1's capability of RIDD, and showing a physiological function of such activity in worms, we think that ER stress response is not limited to directly protecting cells from stresses but can contribute to neuronal control of physiological processes in animals.