Cellular homeostasis and healthy ageing: protein folding, redox and metabolism

在细胞水平上，健康的衰老取决于自身平衡机制的恢复能力。在这方面，调节错误折叠蛋白清除（蛋白质静止）、氧化还原反应、代谢途径和一般损伤反应的途径显得很重要，并且可能存在联系。在组织中，这些过程的崩溃表现为纤维化或瘢痕。我打算探究他们之间的关系。我建议通过使用CRISPR编辑启动子区域来产生一对上皮细胞系，这些细胞系仅在与单一黏液蛋白MUC5B表达相关的蛋白抑制挑战程度上存在差异。为了正确折叠，这种蛋白质需要在六个半胱氨酸残基之间正确形成三对分子内二硫键。这需要大量参与内质网（ER）内氧化还原平衡和蛋白质折叠的调节机制。它在上皮细胞中的过度表达与内质网应激以及组织中的功能失调损伤和瘢痕反应有关。因此，我将使用我的细胞模型来关联蛋白质折叠、氧化还原和代谢重编程中的稳态挑战水平。我进一步建议通过调节内质网应激、氧化还原和线粒体功能来评估这些反应与上皮细胞和间充质细胞之间促纤维化串音的调节相关的程度。

At a cellular level, healthy ageing depends upon the resilience of homoeostatic mechanisms. Pathways regulating clearance of misfolded proteins (proteostasis), redox responses, metabolic pathways and general injury responses appear important in this regard and may be linked. In tissue, collapse of these processes manifests as fibrosis or scarring. I plan to probe their relationships with each other. I propose to do this by using CRISPR editing of a promoter region to generate pairs of epithelial cell lines differing only in the degree of proteostatic challenge relating to expression of a single mucin protein, MUC5B. To fold properly this protein requires correct formation of three pairs of intramolecular disulphide bonds between six cysteine residues. This requires considerable engagement of the regulatory machinery of redox homoeostasis and protein folding within the endoplasmic reticulum (ER). Its overexpression in epithelial cells is associated with ER stress, and with dysfunctional injury and scarring responses in tissue. I will therefore use my cell models to correlate levels of homoeostatic challenge in protein folding, redox, and metabolic reprogramming. I further propose to assess the degree to which these responses relate to regulation of pro-fibrotic cross-talk between epithelial and mesenchymal cells by modulating ER stress, redox, and mitochondrial function.