Abstract
The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding or aggregation and the cellular proteostasis network that monitors and maintains proteome health is poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear. Because aggregation in cells is rapid, nonlinear, and asynchronous, it has not been possible to deconvolve these kinetically linked processes to determine the earliest cellular responses to misfolded proteins. Upon removal of the synthetic, biologically inert ligand shield-1 (S1), AgDD, an engineered variant FK506-binding protein (FKBP1A), rapidly (t½ ∼5 min) unfolds and self-associates, forming detergent-insoluble, microscopic cytoplasmic aggregates. Using global diglycine-capture (K-GG) proteomics, we found here that this solubility transition is associated with immediate increases in ubiquitylation of AgDD itself, along with that of endogenous proteins that are components of the ribosome and the 26S proteasome. We also found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1). We conclude that these proteasomal responses are due to AgDD protein misfolding and not to the presence of detergent-insoluble aggregates.
Highlights
The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases
We found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1)
Both DD and AgDD are conjugated to superfolder GFP, are monomeric, and are uniformly distributed in the cell when bound to their ligand, S1 [9, 10]
Summary
Ubiquitylation of key PN nodes in response to acute misfolding of a single protein To study the earliest global changes to the PN that occur in response to acute protein folding stress, we exploited the ability of AgDD to unfold and aggregate following acute removal of the stabilizing ligand, S1 [9]. The 42 sites exhibiting statistically significant increased ubiquitylation in response to S1 w/o are strikingly enriched in functionally related PN proteins, including chaperones, proteasome subunits, ribosomal proteins, and ribosome biogenesis factors, suggesting that acute unfolding and aggregation of a single abundant protein leads to rapid (Յ10-min) Ub modification of both protein degradation and protein synthesis machinery. A single site in RPN10 (PSMD4, Lys-40) exhibited a modest (1.5-fold) increase in ubiquitylation, reaching statistical significance only at 10 min following S1 w/o These data reveal that acute unfolding and/or aggregation of a single highly expressed cytosolic protein leads to immediate and progressive increases in ubiquitylation of key nodes in the PN network, the ribosome and the 26S proteasome. Nearly stoichiometric modification of RPN13 through in vitro ubiquitylation of isolated proteasomes has been reported to impair substrate binding and degradation [23, 24], it is unclear whether those effects are entirely due to modification ggggReporter Ion Intensity
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