Protein Folding Modulation in Cells Subject to Differentiation and Stress

Cytomimetic media are used to mimic the physicochemical properties of the cellular milieu in an in vitro experiment. The motivation is that compared to entire cells, they can be used efficiently in combination with a broad range of experimental techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data must include the study of specific biomolecular reactions in different cell types, different compartments of a single cells and different cellular conditions. In previous studies, model systems such as cancer cell lines, bacteria or oocytes were used. Here we studied how the environment of cells that undergo neuronal differentiation or proteostasis stress modulates the protein folding equilibrium. We found that NGF induced differentiation leads to a decrease of the melting temperature and a change of the folding mechanism. Proteomic changes that occur upon differentiation could explain this effect, however, we found that the crowding effect remained unchanged. Using MG132, a common proteasome inhibitor and inducer of the unfolded protein response, we show that changes to the quality control machinery modulate the folding equilibrium, leading to protein destabilization at prolonged stress exposure. Our study explores the range of protein folding modulation within cells subject to differentiation or stress that must be encountered in the development of cytomimetic media.