Characterization of NFE2L1-616, an isoform of nuclear factor-erythroid-2 related transcription factor-1 that activates antioxidant response element-regulated genes.

The NFE2L1 transcription factor (aka Nrf1) is a basic leucine zipper protein that performs a critical role in the cellular stress response pathway. Here, we characterized a novel variant of NFE2L1 referred to as NFE2L1-616. The transcript encoding NFE2L1-616 is derived from an intronic promoter, and it has a distinct first exon than other reported full-length NFE2L1 isoforms. The NFE2L1-616 protein constitutively localizes in the nucleus as it lacks the N-terminal amino acid residues that targets other full-length NFE2L1 isoforms to the endoplasmic reticulum. The expression level of NFE2L1-616 is lower than other NFE2L1 isoforms. It is widely expressed across different cell lines and tissues that were examined. NFE2L1-616 showed strong transcriptional activity driving luciferase reporter expression from a promoter containing antioxidant response element. Together, the results suggest that NFE2L1-616 variant can function as a positive regulator in the transcriptional regulation of NFE2L1 responsive genes.

A novel nonsense variant in the NFE2L1 transcription factor in a patient with developmental delay, hypotonia, genital anomalies, and failure to thrive.

The NFE2L1 transcription factor (also known as Nrf1 for nuclear factor erythroid 2-related factor-1) is a broadly expressed basic leucine zipper protein that performs a critical role in the cellular stress response pathway. Here, we identified a heterozygous nonsense mutation located in the last exon of the gene that terminates translation prematurely, resulting in the production of a truncated peptide devoid of the carboxyl-terminal region containing the DNA-binding and leucine-zipper dimerization interface of the protein. Variant derivatives were well expressed in vitro, and they inhibited the transactivation function of wild-type proteins in luciferase reporter assays. Our studies suggest that this dominant-negative effect of truncated variants is through the formation of inactive heterodimers with wild-type proteins preventing the expression of its target genes. These findings suggest the potential role of diminished NFE2L1 function as an explanation for the developmental delay, hypotonia, hypospadias, bifid scrotum, and failure to thrive observed in the patient.

The deubiquitinating enzyme USP7 regulates the transcription factor Nrf1 by modulating its stability in response to toxic metal exposure.

The nuclear factor E2-related factor 1 (Nrf1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of Nrf1 proteins, but the regulatory enzymes that act on Nrf1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of Nrf1 activity. We found that USP7 interacts with Nrf1a and TCF11-the two long protein isoforms of Nrf1. Expression of wildtype USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and Nrf1a, leading to their increased stability and increased transactivation of reporter gene expression by TCF11 and Nrf1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and Nrf1a and reduction of their steady state levels. Loss of USP7 function attenuated the induction of Nrf1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of Nrf1 protein to induce gene expression in response to toxic metal exposure.

Nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) is regulated by O-GlcNAc transferase.

The Nrf1 (Nuclear factor E2-related factor 1) transcription factor performs a critical role in regulating cellular homeostasis. Using a proteomic approach, we identified Host Cell Factor-1 (HCF1), a co-regulator of transcription, and O-GlcNAc transferase (OGT), the enzyme that mediates protein O-GlcNAcylation, as cellular partners of Nrf1a, an isoform of Nrf1. Nrf1a directly interacts with HCF1 through the HCF1 binding motif (HBM), while interaction with OGT is mediated through HCF1. Overexpression of HCF1 and OGT leads to increased Nrf1a protein stability. Addition of O-GlcNAc decreases ubiquitination and degradation of Nrf1a. Transcriptional activation by Nrf1a is increased by OGT overexpression and treatment with PUGNAc. Together, these data suggest that OGT can act as a regulator of Nrf1a.

Use of and interests in complementary and alternative medicine by Hispanic patients of a community health center.

INTRODUCTION: We evaluated complementary and alternative medicine (CAM) use among a medically underserved, predominately Hispanic community at the University of California Irvine Family Health Center, a federally qualified health center. METHODS: A cross-sectional, anonymous survey assessed patient use of, interest in, and communication preferences concerning CAM. RESULTS: The 150 respondents primarily self-identified as Hispanic (74%), were born outside the United States (55%), were medically insured (56%), and had a high school education or less (55%). Of these respondents, 63% used at least 1 type of CAM; the most commonly used were: vitamins/supplements (32%), herbal medicine (29%), dietary/nutritional therapy (26%), massage (24%), meditation/relaxation (15%) and chiropractic (11%). Therapies that patients most desired to see provided at the clinic included massage, healthier cooking, guidance on herbs/supplements, and diet/nutrition. Among respondents, 61% were comfortable disclosing CAM use to physicians, 58% agreed physicians should have basic knowledge of CAM, and 47% desired that physicians ask about CAM use. CONCLUSIONS: Results demonstrate that CAM use is common among patients, and a large proportion of patients have interest in accessing CAM through their primary care clinic. Patients recognize the importance of communicating CAM use with their providers and seem receptive to discussing such topics.

Induction of Herpud1 expression by ER stress is regulated by Nrf1.

Herpud1 is an ER-localized protein that contributes to endoplasmic reticulum (ER) homeostasis by participating in the ER-associated protein degradation pathway. The Nrf1 transcription factor is important in cellular stress pathways. We show that loss of Nrf1 function results in decreased Herpud1 expression in cells and liver tissues. Expression of Herpud1 increases in response to ER stress, but not in Nrf1 knockout cells. Transactivation studies show that Nrf1 acts through antioxidant response elements located in the Herpud1 promoter, and chromatin immunoprecipitation demonstrates that Herpud1 is a direct Nrf1 target gene. These results indicate that Nrf1 is a transcriptional activator of Herpud1 expression during ER stress, and they suggest Nrf1 is a key player in the regulation of the ER stress response in cells.

Nuclear factor-erythroid 2-related factor 1 regulates expression of proteasome genes in hepatocytes and protects against endoplasmic reticulum stress and steatosis in mice.

The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap 'n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.