Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines

Abstract

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

Keywords: NFE2L1; NGLY1deficiency; NRF1; autophagy; deglycosylation.

Figure 1

Workflow for the application of multi-omic data to drug screening in NGLY1 KD K562 cell lines.

Figure 2

Characterization of NGLY1-deficient K562 lines. (A) Western blot analysis of K562 cell lines used in this paper. (B) Flow readout and gating for the analysis of K562 cell lines used in this paper. Data were used to calculate geometric means for the Venus to mCherry ratio. (C) Average geometric mean of the Venus to mCherry signal for all lines used in the paper. (D) Dose response of NGLY1-deficient K562 lines to Bortezomib. 95% confidence interval shown as shading on the graph.

Figure 3

Overlapping correlative analysis of transcripts and proteins detected in NGLY1-deficient K562 cell lines. (A) Fold changes of transcripts and proteins differentially expressed in K562 NGLY1-deficient lines. Colors, as described in the key, correspond to the dataset in which they were found to be differentially expressed. HGNC Gene symbols have been labeled for the most significantly differentially expressed, as determined by a combination of p-value and fold change. (B) Highlight of proteasome subunit gene transcript expression in K562 NGLY1-deficient lines, this gene set (colored in blue) displays a shift toward lower expression in NGLY1-deficient lines. Red points depict the transcripts that were called differentially expressed only on the transcript level. Gray points are all expressed genes. (C) Proteasome subunit genes were identified as significantly enriched downregulated transcripts. X-axis is the number of genes found in the GO category. Numerals after the bar graph represent the ratio of significant genes to total expressed genes in that GO category.

Figure 4

Correction of NGLY1-deficient phenotypes in K562 cell lines by exogenous expression of a DYK-tagged NGLY1 protein. (A) Western blot analysis of NGLY1-deficient K562 cell line's expression of plasmid-based N- or C-terminally tagged NGLY1. Antibodies probed are labeled along the left side of the figure along with corresponding kDa mol. weight markers. The same protein lysates were loaded on two gels. (B) FACS analysis of NGLY1-deficient K562 cell line's expression of plasmid-based N- or C-terminally tagged NGLY1. Results were quantified, averaged, and graphed. (C) Immunofluorescent staining of NGLY1-deficient K562 cell lines with DAPI (blue) and Proteostat (red). (D) Quantification of Proteostat staining of NGLY1-deficient K562 cells by flow cytometry analysis. Rescued lines include both C- and N-terminally tagged NGLY1.

Figure 5

Drug treatment of NGLY1-deficient and WT K562 cell lines. (A) Treatment of WT K562 cells with 48 compounds, plotted by assay and concentration to visualize compounds that were not toxic but still inhibited ddVenus fluorescence. Each point represents a single concentration and are labelled by compound if they decreased ATPlite signal by more than 50% or if they reduced ddVenus signal to the level of the NGLY1 KD line control. (B) Dose response curve for NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells, exemplative of a positively confirmed hit from the screen. (C) ATP measurement of the dose response curve for NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells. (D) Western blot analysis of RTA∆-V5 expression levels after NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells for 5 hr and 24 hr treatment at 15 µM for PAC-1 and 0.5 µM for NVP-BEZ235. Exemplative blot from 3 repeated experiments. (E) Western blot analysis of autophagic flux due to NVP-BEZ235 and PAC-1. Time course treatment of WT K562 cells in the presence of compound. (F) Fluorescent signal of Venus and ddVenus at multiple time points in WT and KD lines.