Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways

Abstract

A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Keywords: protein aggregates; protein quality control; protein turnover; ubiquitin-proteasome system.

Fig. 1.

agDD-GFP forms amorphous aggregate structures. (A) Tomogram of HEK293 agDD-GFP cells 10 min after S1 washout. (B) Tomogram of HEK293 agDD-GFP cells 6 h after S1 washout. Selected cellular structures are annotated. (Scale bar, 200 nm.) Agg, Aggregate; ER, endoplasmic reticulum; Endo, endosome; Mito, mitochondria; Autolys, autolysosome; Autoph, autophagosome.

Fig. 2.

agDD-GFP aggregates are degraded by the ubiquitin–proteasome system. (A) Histograms of agDD-GFP fluorescence levels in HEK293T and HEK293T FIP200^−/− cells post-S1 washout measured by flow cytometry. (B) Live cell confocal imaging of HeLa^dCas9 agDD-GFP cell line post-S1 washout. (C) Histograms of agDD-GFP florescence levels in HeLa^dCas9 cell line post-S1 washout measured by flow cytometry (Left). Histograms of agDD-GFP florescence levels in HeLa^dCas9 cell line post-S1 washout in the presence of inhibitors measured by flow cytometry BTZ (bortozomib): 1 μM; SAR405 (VPS34 inhibitor): 1 μM (Right). (D) Histograms of agDD-GFP florescence levels in HeLa^dCas9 agDD-GFP^LOW and agDD-GFP^HIGH cells post-S1 washout measured by flow cytometry.

Fig. 3.

agDD-GFP degradation kinetics is dependent on misfolded protein burden. (A) Confocal microscopy max intensity projection of HeLa^dCas9 agDD-GFP^LOW and agDD-GFP^HIGH cells at various times post-S1 washout. agDD-GFP signal is shown in green, and nucleus is in cyan. (B) Quantification of agDD-GFP aggregate number per cell at various times post-S1 washout. The line represents mean and shaded area is SD. (C) Quantification of average agDD-GFP aggregate size in μm². The line represents mean and shaded area is SD.

Fig. 4.

UBE3C is necessary for acute aggregate clearance. (A) Workflow for gene knock down in HeLa^dCas9 cell lines that contain agDD-GFP. First, cells are infected with a guide against the indicated gene (e.g., UBE3C). After selection, S1 is washed out resulting in agDD-GFP aggregation and degradation, which is measured via GFP fluorescence by flow cytometry. (B) Histograms of agDD-GFP fluorescence levels in HeLa^dCas9 agDD-GFP^LOW cells post-S1 washout in control cells (Left) and UBE3C knockdown (KD) cells (Right) measured by flow cytometry. (C) Histograms of agDD-GFP fluorescence levels in HeLa^dCas9 agDD-GFP^HIGH post-S1 washout in control cells (Left) and UBE3C knockdown cells (Right) measured by flow cytometry. (D) Western blot of purified proteasomes from HEK293T agDD-GFP and HEK293T agDD-GFP cells with endogenous mutations in RPN13 K21R and K34R post-S1 washout or BTZ treatment. (E) Histograms of agDD-GFP fluorescence levels in HEK293T agDD-GFP cells (Left) and RPN13^K21R;K34R mutations (Right) post-S1 washout measured by flow cytometry. (F) Histograms of agDD-GFP fluorescence levels in HEK293T agDD-GFP^HIGH cells (Left) and agDD-GFP^HIGH cells with RPN13^K21R;K34R mutations (Right) post-S1 washout measured by flow cytometry.

Fig. 5.

NRF1 is activated by sustained agDD-GFP aggregation. (A) Relative mRNA levels for proteasome subunits measured in HeLa^dCas9agDD-GFP^HIGH cells after S1 washout or BTZ treatment (10 nM). *P > 0.05, ***P > 0.01 using a student t test. (B) Western blot of NRF1 protein after S1 washout or BTZ treatment (10 nM). Glycans are removed from NRF1 using EndoH (Right). (C) Confocal microscopy of HeLa^dCas9 agDD-GFP cell line post S1 washout or BTZ treatment (10 nM). Nuclei are shown in cyan, NRF1 in magenta, and agDD-GFP in green. (D) Quantification of panel C: NRF1 intensity per cell. *P-value < 0.05 using a student t test. (E) Quantification of panel C: nuclear NRF1 verses total signal. *P-value < 0.05 using a student t test.

Fig. 6.

NRF1 modulates aggregation clearance. (A) Log₂ fold change of GFP fluorescence in HeLa^dCas9 agDD-GFP^HIGH cells with NRF1 or DDI2 knockdown post-S1 washout. Error bars show SD. * indicates p-value < 0.05 using a Dunnett’s test. NRF1 vs control P = 0.013, DDI2 vs control P = 0.020. (B) Log₂ fold change of GFP fluorescence in HeLa^dCas9 agDD-GFP^HIGH cells that are overexpressing wild-type or 18ND mutant NRF1 post-S1 washout. Error bars show SD. * indicates p-value < 0.05 using a Dunnett’s test. WT NRF1 vs control: 5 h; P = 0.00477, 16 h; 0.0345. 18ND NRF1 vs control: 5 h; P = 0.005, 16 h; 0.0482. (C) Model of protein quality control pathways that respond to widespread protein aggregation. At low levels of misfolded proteins or small aggregates/oligomers, UBE3C ubiquitylates substrates to promote efficient proteasomal degradation. When levels of misfolded proteins are high, larger inclusions are formed that are increasingly resistant to acute UBE3C-mediated degradation, resulting in activation of NRF1 and downstream proteasome subunit transcription.