It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.

Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This “bounce-back” response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

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.