Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes

J Biol Chem. 2019 Nov 8;294(45):16511-16524. doi: 10.1074/jbc.RA119.009654. Epub 2019 Aug 2.


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 (t ½ ∼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.

Keywords: ADRM1; E3 ubiquitin ligase; RPN13; UBE3C; proteasome; protein aggregation; protein misfolding; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Morpholines / chemistry
  • Morpholines / metabolism
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Aggregates
  • Protein Subunits / metabolism
  • Protein Unfolding
  • Proteomics
  • Proteostasis
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Tacrolimus Binding Proteins / genetics
  • Tacrolimus Binding Proteins / metabolism
  • Ubiquitin-Protein Ligases / antagonists & inhibitors
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination


  • ADRM1 protein, human
  • FKBP1A protein, human
  • Intracellular Signaling Peptides and Proteins
  • Morpholines
  • Protein Aggregates
  • Protein Subunits
  • RNA, Small Interfering
  • Shield-1 compound
  • UBE3C protein, human
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease
  • Tacrolimus Binding Proteins