N-glycanase 1 Research Articles

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1−/− rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1−/− rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1−/− rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1−/− rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1−/− rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

Abstract 3048: Targeting protein deglycosylation as a newly identified vulnerable point in melanoma

Abstract As an enzyme that removes N-glycans from glycopeptides, N-glycanase 1 (NGLY1) deglycosylates denatured glycoproteins and allows proteasome-mediated protein degradation to efficiently occur. Although NGLY1 is known for this pivotal function, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. Here, we examined how NGLY1 expression affects viability, tumor growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumors. NGLY1 knockdown caused melanoma cell death and tumor growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signaling-associated apoptosis and cytokine surges, which synergize with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy with the opportunity for a broad therapeutic window. Citation Format: Victor J.T. Lin, Ashwini Zolekar, Nigam M. Mishra, Yin Ying Ho, Hamed S. Hayatshahi, Abhishek Parab, Rohit Sampat, Xiaoyan Liao, Peter Hoffmann, Jin Liu, Kyle A. Emmitte, Yu-Chieh Wang. Targeting protein deglycosylation as a newly identified vulnerable point in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3048.

N-Glycanase 1 Regulates Aquaporins Independent of Its Enzymatic Activity

Patients with pathogenic mutations in NGLY1 cannot make tears (alacrima), have global developmental delay and liver dysfunction. Traditionally, N-glycanase 1 (NGLY1) cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins prior to their proteasomal degradation. We found that Ngly1-null mouse embryonic fibroblasts (MEFs), NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient MEFs swell slower and have reduced aquaporin 1 (Aqp1) mRNA and protein expression. Ngly1 knockdown and overexpression confirmed that Ngly1 regulates Aqp1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 KO cells show reduced AQP11 mRNA, supporting NGLY1 regulates expression of multiple AQPs across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restored normal hypotonic lysis and Aqp1 protein. We show that transcription factor Creb1 regulates Aqp1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient MEFs. These results identify a novel, non-enzymatic, regulatory function of NGLY1 in AQP transcription possibly related to alacrima.

N-glycanase NGLY1 regulates mitochondrial homeostasis and inflammation through NRF1.

Mutations in the NGLY1 (N-glycanase 1) gene, encoding an evolutionarily conserved deglycosylation enzyme, are associated with a rare congenital disorder leading to global developmental delay and neurological abnormalities. The molecular mechanism of the NGLY1 disease and its function in tissue and immune homeostasis remain unknown. Here, we find that NGLY1-deficient human and mouse cells chronically activate cytosolic nucleic acid-sensing pathways, leading to elevated interferon gene signature. We also find that cellular clearance of damaged mitochondria by mitophagy is impaired in the absence of NGLY1, resulting in severely fragmented mitochondria and activation of cGAS-STING as well as MDA5-MAVS pathways. Furthermore, we show that NGLY1 regulates mitochondrial homeostasis through transcriptional factor NRF1. Remarkably, pharmacological activation of a homologous but nonglycosylated transcriptional factor NRF2 restores mitochondrial homeostasis and suppresses immune gene activation in NGLY1-deficient cells. Together, our findings reveal novel functions of the NGLY1-NRF1 pathway in mitochondrial homeostasis and inflammation and uncover an unexpected therapeutic strategy using pharmacological activators of NRF2 for treating mitochondrial and immune dysregulation.

Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency.

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl. We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome “bounce-back” response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.

Beyond ERAD: N‐glycanase will bring you to tears

Patients with mutations in NGLY1 cannot make tears (alacrima), have global developmental delay, movement disorder and liver dysfunction. N‐glycanase 1 (NGLY1) de‐glycosylates misfolded N‐glycosylated proteins in the cytoplasm as part of the ERAD pathway prior to their proteasomal degradation. Surprisingly, NGLY1‐deficient patient cells do not accumulate cytoplasmic misfolded N‐glycoproteins, suggesting a more complex function. Interestingly, new studies show NGLY1 is needed for transcription factor NFE2L1/NRF1 activation and normal mitochondrial function.Using WT and NGLY1‐deficient mouse embryonic fibroblasts (MEFs) we found that NGLY1‐deficient cells were resistant to hypotonic lysis compared to WT. The same was seen in NGLY1‐patient fibroblasts. Additionally, MEFs deficient in both NGLY1 and ENGase (another deglycosylation enzyme) were even more resistant to hypotonic lysis compared to NGLY1‐deficient cells. Water influx and cell swelling precedes cell lysis. We found that NGLY1‐deficient MEFs swell slower than WT MEFs. Since aquaporins (AQP) transport water, we hypothesized that AQP levels might be disrupted in NGLY1‐deficient cells. We found both AQP1 mRNA and protein were reduced in NGLY1‐deficient MEFs. shRNA knockdown of AQP1 in WT MEFs decreased hypotonic lysis, suggesting AQP1 is associated with hypotonic lysis. NGLY1 shRNA and CRISPR studies confirmed that NGLY1 regulates AQP1 levels and hypotonic cell lysis. Preliminary studies show that complementing NGLY1‐deficient MEFs with NGLY1 increases AQP1 and restores hypotonic lysis. Current efforts are directed towards understanding whether NGLY1 enzyme activity is necessary for AQP regulation. 13 types of AQP are expressed in different cell types. Our preliminary data suggest that AQP11 levels are disrupted in NGLY1‐deficient patient cells and CRISPR cells, suggesting that NGLY1 might regulate different types of AQP.We have identified a novel function of NGLY1, i.e. to directly/indirectly regulate AQPs. This finding may relate to NGLY1‐deficient patients' inability to make tears. Future efforts will try to identify NGLY1‐dependent transcription factors responsible for regulating AQPs. This work is supported by the Bertrand Might Research Fund and NGLY1.org.Support or Funding InformationThis work is supported by the Bertrand Might Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A New Fluorogenic Probe for the Detection of endo-β-N-Acetylglucosaminidase.

We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-β-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.

Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches.

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1.

Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme.

New perspectives on the mutated NGLY1 enigma

The enzyme N-glycanase 1 (NGLY1) is considered a component of the endoplasmic reticulum-associated degradation (ERAD) machinery and clinical manifestations of its dysfunction include global developmental delay, a movement disorder, peripheral neuropathy, liver disorders, microcephaly, diminished reflexes and seizures. Although several mutations in NGLY1 have been identified, the relation between the defected protein and the above described pathologies is yet unknown. We hypothesised that NGLY1 failure to degrade certain proteins may result in their accumulation and overexpression and used a systems biology approach to identify proteins that may be affected by NGLY1 deficiency. Genes that interact with the NGLY1 gene according to BioGRID database of physical and genetic interactions were analysed with STRING Protein–Protein interaction database. Network analysis identified FAF1 (Fas-Associated Factor 1), an apoptosis-potentiating protein, as a possible degradation substrate of NGLY1. Examination of normal tissue microarrays demonstrated that FAF1-to-NGLY1 ratio is maximal (more than 3:1) in skeletal muscle and brain tissues microarrays. This evidence may explain the pathologies in brain and muscle tissues of patients with mutated NGLY1. To test this hypothesis, laboratory studies that will assess if FAF1 protein is overexpressed in tissues of patients with mutated NGLY1 are required.

NGLY1 mutation causes neuromotor impairment, intellectual disability, and neuropathy

N-glycanase 1 (NGLY1) is a conserved enzyme that is responsible for the deglycosylation of misfolded N-glycosylated proteins in the cytoplasm prior to their proteasome-mediated degradation. Disruption of this degradation process has been associated with various neurologic diseases including amyotrophic lateral sclerosis and Parkinson's disease. Here, we describe two siblings with neuromotor impairment, apparent intellectual disability, corneal opacities, and neuropathy who were found to possess a novel homozygous frame-shift mutation due to a 4 base pair deletion in NGLY1 (c.1533_1536delTCAA, p.Asn511LysfsX51). We hypothesize that this mutation likely limits the capability of neuronal cells to respond to stress due to accumulation of misfolded proteins, thereby impairing their survival and resulting in progressive loss of neurological function.

Doubling of optic disc

<h3>Abstract</h3> NGLY1 (N-glycanase 1) deficiency is a rare congenital recessive disorder caused by a mutation in the <i>NGLY1</i> gene, which encodes the cytosol enzyme N-glycanase 1. The NGLY1 protein catalyzes...