Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics

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.

Figure 1.. SG Protein Ubiquitylation Is Largely Unaffected by NaAsO₂ Treatment

(A) Immunoblot of phospho-eIF2a, ubiquitin, and tubulin in whole-cell extracts from HeLa cells treated with NaAsO₂ as indicated. s and l denote short and long exposures, respectively. (B) Log₂ heavy-to-light ratios (log₂ H/L) for all diGLY-modified peptides (top) and total proteins (bottom). Heavy-labeled cells were treated with NaAsO₂ as indicated. (C) The fraction of all quantified diGLY peptides with >2-fold change in abundance upon NaAsO₂ treatment or washout. (D) Log₂ H/L corresponding to diGLY-modified peptides from ubiquitin. The individual ubiquitin modified lysine that was quantified is indicated. Error bars denote SEM of multiple quantification events for a given peptide. (E) The median log₂ H/L of all diGLY-modified peptides quantified from known SG proteins. Error bars denote SEM for all diGLY quantification events observed on known SG proteins. NS, not significant. See also Figure S1 and Tables S1 and S2.

Figure 2.. The Ubiquitin-Activating Enzyme Inhibitor TAK-243 Rapidly Ablates Ubiquitin Conjugates in Cells

(A and B) Immunoblot of Nedd8, ubiquitin, and tubulin in whole-cell extracts from HCT116 cells treated with Ub-E1i or N8-E1i alone (A and B) or in combination with MG132 (A) as indicated. s and l denote short and long exposures, respectively. (C) Log₂ H/L for all diGLY-modified peptides (top) and total proteins (bottom). Heavy-labeled cells were treated with Ub-E1i (1 μM) with or without MG132 (10 μM) for 4 h. (D) The fraction of all quantified diGLY-modified peptides that increased, decreased, or remained unchanged upon Ub-E1i treatment with or without proteasome inhibition. (E) Log₂ H/L corresponding to diGLY-modified peptides from ubiquitin after Ub-E1i treatment alone. The individual ubiquitin-modified lysine that was quantified is indicated. Error bars denote SEM of multiple quantification events for a given peptide. (F) Log₂ H/L of all diGLY-modified peptides quantified from known SG proteins. Heavy-labeled cells were treated with Ub-E1i (1 μM) with or without MG132 (10 μM) for 4 h. See also Tables S2 and S3.

Figure 3.. SG Dynamics Are Unaffected by Inhibition of Protein Ubiquitylation or Neddylation

(A, C, E, and G) Quantification of stress granule (SG) dynamics in 293T G3BP1-GFP (A and C) and HeLa (E and G) cells during SG assembly (A and E) and disassembly (C and G). Cells in multi-well plates were treated with inhibitors and NaAsO₂ as indicated, with or without subsequent washout. Following fixation at the indicated time points, HeLa cells were immunostained with an antibody against G3BP1, and plates were imaged using an automated image acquisition system. SG and nuclear area were quantified using custom image analysis scripts, and error bars denote SD. A minimum of ~25,000 cells were analyzed for each cell type, condition, or time point. (B, D, F, and H) Representative images for indicated time points and treatments quantified in (A),(C), (E), and (G), respectively. Scale bars, 20 μm. See also Figures S2 and S3.

Figure 4.. Unconjugated Ubiquitin Localizes to SGs in a UAE-Independent Manner

(A–E) Immunofluorescence staining of HeLa cells treated with NaAsO₂ (250 μM) for 0’, 45’, or 120’ prior to fixation (A) or pretreated with DMSO or UbE1i (1 μ M) for 90’, followed by treatment with NaAsO₂ (250 mM) for 120’ prior to fixation (B–E). Cells were stained with antibodies against G3BP1 (A–E), pan-ubiquitin (A), polyubiquitin (B and D), centrosome marker pericentrin (D), proteasome subunits (D), VCP/p97 (E), SQSTM/p62 (E), HDAC6 (E), UBAP2L (E), or TRIM 25 (E). (C) Cells stably expressing either mCherry-tagged wild-type ubiquitin (mCh-Ub-WT) or mCherry-tagged ubiquitin, in which all internal lysine residues were mutated to arginine and the C-terminal diglycine residues were removed (mCh-Ub-K0DGG [K0dGG]) were stained with antibodies against G3BP1. G3BP1-positive SGs are indicated by arrows (both red and white). G3BP1-negative perinuclear foci are indicated by solid yellow arrowheads. G3BP1-negative punctate signal for VCP/p97 and SQSTM/p62 is indicated by blue open arrowheads. Images from Ub-E1i-treated cells in all panels were taken at the same exposure time and acquisition settings in the ubiquitin channel as those for images from DMSO-treated cells. Nuclei were stained using DAPI. Scale bars, 20 μ m in all panels. See also Figure S4.