No abstract

An extensive network of chaperones and other proteins maintain protein homeostasis and guard against inappropriate protein aggregation that is a hallmark of neurodegenerative diseases. Using a fluorescence resonance energy-based biosensor that simultaneously reports on intact cellular chaperone holdase activity and detrimental aggregation propensity, we investigated the buffering capacity of the systems managing protein homeostasis in the nucleus of the human cell line HEK293 compared to the cytosol. We found that the nucleus showed lower net holdase activity and reduced capacity to suppress protein aggregation, suggesting that the nuclear quality control resources are less effective compared to those in the cytosol. Aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to deplete cytosolic chaperone supply by depleting holdase activity. Unexpectedly, the same stress increased holdase activity in the nucleus suggesting that proteostasis stress can trigger a rebalance of chaperone supply in different subcellular compartments. Collectively the findings suggest the cytosol has more capacity to manage imbalances in proteome foldedness than the nucleus, but chaperone supply can be redirected into the nucleus under conditions of proteostasis stress caused by cytosolic protein aggregation.

No abstract

This protocol describes a method for studying the proteostasis capacity of cells using bait protein biosensors based on the model protein barnase. Quality control machinery in the cell engages with barnase, and modifies its foldedness and aggregation. Through fluorescent tags on the barnase bait, flow cytometry FRET measurements provide a readout of foldedness and aggregation, enabling comparison of quality control capacity under different conditions. The method involves transient transfection with the barnase bait, data collection via flow cytometry, and data analysis. The procedure takes approximately one week. An additional procedure enables data to be fit to a mechanistic model for better quantitation. These additional steps require protein expression and purification, as well as microscopy for determining cell volume, expected to add an additional 1-2 weeks.

No abstract