Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-β-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Cancer cells subvert multiple properties of normal cells, including escaping strict cell cycle regulation, gaining resistance to cell death, and remodeling the tumor microenvironment. The hallmarks of cancer have recently been updated and summarized. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also named NRF1) belongs to the cap\'n\'collar (CNC) basic-region leucine zipper (bZIP) family. It acts as a transcription factor and is indispensable for maintaining both cellular homoeostasis and organ integrity during development and growth, as well as adaptive responses to pathophysiological stressors. In addition, NFE2L1 mediates the proteasome bounce-back effect in the clinical proteasome inhibitor therapy of neuroblastoma, multiple myeloma, and triple-negative breast cancer, which quickly induces proteasome inhibitor resistance. Recent studies have shown that NFE2L1 mediates cell proliferation and metabolic reprogramming in various cancer cell lines. We combined the framework provided by \"hallmarks of cancer\" with recent research on NFE2L1 to summarize the role and mechanism of NFE2L1 in cancer. These ongoing efforts aim to contribute to the development of potential novel cancer therapies that target the NFE2L1 pathway and its activity.

Ferroptosis is an iron-dependent cell death mechanism that may be important to prevent tumor formation and useful as a target for new cancer therapies. Transcriptional networks play a crucial role in shaping ferroptosis sensitivity by regulating the expression of transporters, metabolic enzymes, and other proteins. The Cap\'n\'collar (CNC) protein nuclear factor erythroid 2 like 2 (NFE2L2, also known as NRF2) is a key regulator of ferroptosis in many cells and contexts. Emerging evidence indicates that the related CNC family members BTB and CNC homology 1 (BACH1) and nuclear factor erythroid 2 like 1 (NFE2L1) also have non-redundant roles in ferroptosis regulation. Here, we comprehensively review the role of CNC transcription factors in governing cellular sensitivity to ferroptosis. We describe how CNC family members regulate ferroptosis sensitivity through modulation of iron, lipid, and redox metabolism. We also use examples of ferroptosis regulation by CNC proteins to illustrate the flexible and highly context-dependent nature of the ferroptosis mechanism between cells and conditions.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as Nrf1) is a crucial member of the CNC-bZIP subfamily of transcription factors expressed ubiquitously throughout our body. Recent findings have revealed its association with various metabolic processes, encompassing glucose, lipid, and protein metabolism. In the realm of glucose metabolism, NFE2L1 exerts regulatory control by modulating pancreatic β cells and insulin production. It also influences glucose metabolism in liver and the insulin sensitivity of adipose tissue. Regarding lipid metabolism, NFE2L1 governs this process by influencing the expression of specific adipogenic and lipolysis genes in both liver and adipose tissue. Additionally, NFE2L1 regulates specific lipids, such as cholesterol. These involvements underlie various manifestations of NFE2L1 deficiency such as adipocyte hypertrophy, inflammation, and steatohepatitis. In the realm of protein metabolism, NFE2L1 serves as a major transcription factor regulating the 26S proteasome genes expression, which dysfunction has been related with multiple diseases including neurodegenerative diseases, cancers, autoimmune conditions, etc. In this comprehensive review, we summarize the diverse roles that NFE2L1 plays in glucose, lipid, and protein metabolism, as well as its impact on diseases related to these metabolic processes.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs).
RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm.
CONCLUSIONS: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

BACKGROUND: Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown.
METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry.
RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells.
CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.

Oral squamous cell carcinoma (OSCC) is a common head and neck malignancy with increasing mortality and high recurrence. In this work, we aim to explore the functional role of NFE2 like bZIP transcription factor 1 (NFE2L1) in OSCC progression. Based on databases analysis, we found that NFE2L1 was overexpressed in OSCC tumor tissues, and elevated NFE2L1 level induced poor prognosis of OSCC patients. Our results showed that NFE2L1 is upregulated in OSCC cells and overexpression of NFE2L1 promotes cell proliferation, and reduces the sensitivity of OSCC cells to erastin-induced ferroptosis. NFE2L1 upregulation decreased the levels of Fe2+, lipid reactive oxygen species and content of malondialdehyde, and increased the level of the key negative regulator of ferroptosis, GPX4 and SLC7A11. In NFE2L1 suppressed cells, these trends were reversed. Further results of dual luciferase reporter and chromatin immunoprecipitation assays confirmed that NFE2L1 could bind to the promoter of Holliday junction recognition protein (HJURP) to increase the transcriptional activity of HJURP, thus upregulating its expression. Inhibition of HJURP attenuated the proliferation and ferroptosis inhibition in NFE2L1 upregulated cells. In vivo tumorigenicity assay further proved that NFE2L1 promotes OSCC tumor growth. In summary, NFE2L1 restrains ferroptosis by transcriptionally regulating HJURP and participates in the progress of OSCC. Thus, NFE2L1 plays a key role in OSCC development and may be a promising therapeutic target for OSCC.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis (\"proteostasis\") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced \"bounce-back effect\" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

Brown adipose tissue (BAT) is a major site of non-shivering thermogenesis in mammals and plays an important role in energy homeostasis. Nuclear factor-erythroid 2-related factor 1 (NFE2L1, also known as Nrf1), a master regulator of cellular metabolic homeostasis and numerous stress responses, has been found to function as a critical driver in BAT thermogenic adaption to cold or obesity by providing proteometabolic quality control. Our recent studies using adipocyte-specific Nfe2l1 knockout [Nfe2l1(f)-KO] mice demonstrated that NFE2L1-dependent transcription of lipolytic genes is crucial for white adipose tissue (WAT) homeostasis and plasticity. In the present study, we found that Nfe2l1(f)-KO mice develop an age-dependent whitening and shrinking of BAT, with signatures of down-regulation of proteasome, impaired mitochondrial function, reduced thermogenesis, pro-inflammation, and elevated regulatory cell death (RCD). Mechanistic studies revealed that deficiency of Nfe2l1 in brown adipocytes (BAC) primarily results in down-regulation of lipolytic genes, which decelerates lipolysis, making BAC unable to fuel thermogenesis. These changes lead to BAC hypertrophy, inflammation-associated RCD, and consequently cold intolerance. Single-nucleus RNA-sequencing of BAT reveals that deficiency of Nfe2l1 induces significant transcriptomic changes leading to aberrant expression of a variety of genes involved in lipid metabolism, proteasome, mitochondrial stress, inflammatory responses, and inflammation-related RCD in distinct subpopulations of BAC. Taken together, our study demonstrated that NFE2L1 serves as a vital transcriptional regulator that controls the lipid metabolic homeostasis in BAC, which in turn determines the metabolic dynamics, cellular heterogeneity and subsequently cell fates in BAT.