Protein homeostasis (proteostasis) is regulated by an extensive quality control network governing protein synthesis, folding, transport and degradation. When proteostasis becomes unbalanced protein misfolding and aggregation is observed. Critically, inappropriate protein aggregation and proteostasis imbalance are two of the central pathological features of common neurodegenerative diseases including Alzheimer, Parkinson, Huntington, and motor neuron diseases. However, it remains unclear to which proteins in the proteome become preferentially altered in aggregation state in disease and how these patterns relate to mechanisms of proteostasis imbalance.
To investigate this problem, we devised a quantitative proteomic workflow for cell culture samples that have been fractionated into soluble and insoluble fractions after treatment with distinct pharmacological stresses on proteostasis including Hsp90 inhibition, and Hsp70 inhibition, proteasome inhibition, ER stress induction, and oxidative stress. We find that by and large these treatments yield distinct subproteomes that are altered in solubility, which produce molecular signatures for the type of stress invoked. We also compared these findings to the aggregation of the Huntingtin exon 1 protein (Htt) in a Huntington Disease cell model. For this we examined cells expressing mutant Htt that had been first separated into populations lacking Huntingtin aggregates from those with aggregates using our previously developed flow cytometry-based Pulse Shape Analysis. We found soluble mutant Htt induced a cluster of nuclear and mitochondrial proteins to change their solubility. When Htt aggregated, proteins involved in quality control including protein refolding and ER-associated degradation became more insoluble. These data indicated that mutant Htt aggregation led to a cascade of proteome solubility changes that involve a mixture of the stress signatures, notably those associated with ER stress, oxidative stress and impaired proteostasis.