A biosensor to gauge protein homeostasis resilience differences in the nucleus compared to cytosol of mammalian cells

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.

Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

“…While we cannot rule out differences in the sensitivity of the Fluc-EGFP sensor to proteostasis impairments in different cellular compartments, our results support the idea that nuclear and cytoplasmic compartments differ in their capacity to cope with protein aggregation. This is in agreement with studies in yeast and in cell lines describing distinct protein quality control mechanisms operating in the nucleus and cytoplasm (Hageman et al, 2007;Park et al, 2013;Woerner et al, 2016;Enam et al, 2018;Samant et al, 2018;Frottin et al, 2019;den Brave et al, 2020;Raeburn et al, 2021). Although we did not observe any Fluc-EGFP response to mHTT IBs localized in the nucleus, it should be noted that our findings do not exclude deleterious effects of nuclear mHTT, which have been demonstrated previously in cultured cells and animal models (Saudou et al, 1998;Yang et al, 2002;Bae et al, 2006;Gu et al, 2015;Veldman et al, 2015).…”

Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability

“…We next explored the idea that the extra load of unfolded protein upon HS could reinforce partitioning of SOD1 bar mutants into SGs, as pre-existing unfolded proteins remain bound to PQC , and are thus unavailable to be recruited. We determined the unfolded fractions for each construct at 37 and 43 °C from Δ G f °′ at these same temperatures and plotted the change, Δ f U 37–43°C against the different PCs (Figure S5A).…”

Biosensors for Studying Neuronal Proteostasis

“…Cellular compartments and organelles such as the nucleus, mitochondria, and endoplasmic reticulum have evolved distinct proteostasis pathways, and recent studies have highlighted the impact of these organelle-specific mechanisms on cellular proteostasis (Walter and Ron, 2011 ; Kirstein et al, 2015 ; Miller et al, 2015 ; Frakes and Dillin, 2017 ; Shpilka and Haynes, 2018 ; Frottin et al, 2019 ; Moehle et al, 2019 ). Some of the existing proteostasis sensors have already been targeted to different cellular compartments (Winkler et al, 2010 ; Frottin et al, 2019 ; Blumenstock et al, 2021 ; Melo et al, 2021 ; Raeburn et al, 2021 ). A set of spectrally distinct, compartment-specific sensors that could be combined with each other would be a helpful resource for elucidating intra-compartmental crosstalk.…”