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 15 neuromuscular disease. Patients display multi-organ symptoms including developmental delays, 16 movement disorders, seizures, constipation and lack of tear production. NGLY1 is a 17 deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from 18 the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased 19 deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the 20 loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and 21 results in downregulation of proteasomal subunits, consistent with its processing of the 22 transcription factor NFE2L1. We employed the CMap database to predict compounds that can 23 modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the 24 compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, 25 concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in 26 clearing aberrant substrates during NGLY1 deficiency. 27 28 Running Title -Influence of NGLY1 in K562 cells 29 Total Character count -69,046 (including spaces), 58,645 (excluding spaces) 30

NGLY1 knockdown or pharmacological inhibition induces cellular autophagy

“…Similar downregulation at the transcriptional level of proteasomal subunits was also observed following NGLY1 KD in human K562 cells and was interpreted as related to reduced post-translational processing of NFE2L1 to the mature transcription factor NRF1 by NGLY1 (Mueller et al, 2020). Consistent with these observations it was found that NGLY1 disruption sensitizes towards proteasome inhibition by bortezomib in human cells (Mueller et al, 2020) and Drosophila larvae (Rodriguez et al, 2018). The NGLY1 KO mouse model showed the importance of the genetic background towards embryonic lethality and demonstrated that concomitant KO of the Engase gene partially rescued embryonic lethality in C57BL/6 mice (Fujihira et al, 2017).…”

“…The study in the Drosophila model also showed evidence for cnc (fly ortholog of NRF1) dysfunction by transcriptome analysis among downregulated genes (such as those encoding for proteasomal subunits) and an upregulation of the heat shock response (Owings et al, 2018). Similar downregulation at the transcriptional level of proteasomal subunits was also observed following NGLY1 KD in human K562 cells and was interpreted as related to reduced post-translational processing of NFE2L1 to the mature transcription factor NRF1 by NGLY1 (Mueller et al, 2020). Consistent with these observations it was found that NGLY1 disruption sensitizes towards proteasome inhibition by bortezomib in human cells (Mueller et al, 2020) and Drosophila larvae (Rodriguez et al, 2018).…”

“…Recent findings have demonstrated novel cellular functions of NGLY1 in the regulation of aquaporins independent of its enzymatic activity (Tambe et al, 2019) and N-glycanase dependent sequence editing of Nrf-1, leading to the induction of gene expression of proteasomal subunits (Lehrbach et al, 2019;Tomlin et al, 2017;Yang et al, 2018). Recently it has also been shown that knockdown (KD) of NGLY1 in K562 human myelogenous leukemia cells increases sensitivity towards proteasome inhibition and induces complex changes in transcript and protein regulation (Mueller et al, 2020). Despite these important findings there is still limited mechanistic information on the effects of NGLY1 inhibition or disruption in mammalian cells.…”

A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency

“…While perturbations to ERAD often result in ER stress, we have previously reported that there was no functional or transcriptome evidence for ER stress in a Drosophila model of NGLY1 deficiency ( Owings et al, 2018 ). Others have reported no ER stress in NGLY1 -/- human cells, mice, and rats ( Asahina et al, 2020 ; Mueller et al, 2020 ; Tambe et al, 2019 ). However, there is conflicting evidence for ER stress as it was recently reported that ER stress markers were upregulated in NGLY -/- MEFs ( Galeone et al, 2020 ).…”

“…It has been shown that model substrates can be degraded regardless of glycosylation state (Hirsch et al, 2003;Kario, Tirosh, Ploegh, & Navon, 2008). While a recent report showed that ER stress markers were increased in NGLY1 -/-MEFs (Galeone et al, 2020), other experiments such as RNAi knockdown of NGLY1 in Drosophila, (Owings, Lowry, Bi, Might, & Chow, 2018) and loss of NGLY1 function in mouse, rat, and human cells (Asahina et al, 2020;Mueller et al, 2020;Tambe, Ng, & Freeze, 2019) have shown no evidence of ER stress. ER stress is often observed when there are mutations in proteins that are necessary for ERAD due to the accumulation of misfolded proteins in the ER.…”