Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics

Cell Rep. 2019 Apr 30;27(5):1356-1363.e3. doi: 10.1016/j.celrep.2019.04.015.

Abstract

Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics. We show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Critically, inhibiting neither UAE nor NAE significantly affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Using antibodies with varying preference for free ubiquitin or polyubiquitin and fluorescently tagged ubiquitin variants in combination with UAE inhibition, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions.

Keywords: Nedd8; centrosome; neurodegeneration; oxidative stress; proteasome; protein aggregation; protein homeostasis; sodium arsenite; stress granule; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytoplasmic Granules / metabolism*
  • HCT116 Cells
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • NEDD8 Protein / metabolism*
  • Proteasome Endopeptidase Complex / metabolism
  • Stress, Physiological*
  • Ubiquitin-Activating Enzymes / metabolism
  • Ubiquitination*

Substances

  • NEDD8 Protein
  • NEDD8 protein, human
  • Proteasome Endopeptidase Complex
  • Ubiquitin-Activating Enzymes