Glucose Starvation-Induced Rapid Death of Nrf1α-Deficient, but Not Nrf2-Deficient, Hepatoma Cells Results from Its Fatal Defects in the Redox Metabolism Reprogramming

Metabolic reprogramming exists in a variety of cancer cells, with the most relevance to glucose as a source of energy and carbon for survival and proliferation. Of note, Nrf1 was shown to be essential for regulating glycolysis pathway, but it is unknown whether it plays a role in cancer metabolic reprogramming, particularly in response to glucose starvation. Herein, we discover that Nrf1α-/- hepatoma cells are sensitive to rapid death induced by glucose deprivation, such cell death appears to be rescued by Nrf2 interference, but HepG2 (wild-type, WT) or Nrf2-/- cells are roughly unaffected by glucose starvation. Further evidence revealed that Nrf1α-/- cell death is resulted from severe oxidative stress arising from aberrant redox metabolism. Strikingly, altered gluconeogenesis pathway was aggravated by glucose starvation of Nrf1α-/- cells, as also accompanied by weakened pentose phosphate pathway, dysfunction of serine-to-glutathione synthesis, and accumulation of reactive oxygen species (ROS) and damages, such that the intracellular GSH and NADPH were exhausted. These demonstrate that glucose starvation leads to acute death of Nrf1α-/-, rather than Nrf2-/-, cells resulting from its fatal defects in the redox metabolism reprogramming. This is owing to distinct requirements of Nrf1 and Nrf2 for regulating the constructive and inducible expression of key genes involved in redox metabolic reprogramming by glucose deprivation. Altogether, this work substantiates the preventive and therapeutic strategies against Nrf1α-deficient cancer by limiting its glucose and energy demands

A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is a master regulator of antioxidant and/or electrophile response elements (AREs/EpREs)-driven genes involved in homeostasis, detoxification, and adaptation to various stresses. The cytoprotective activity of Nrf2, though being oppositely involved in both cancer prevention and progression, is critically controlled by Keap1 (Kelch-like ECH-associated protein 1), which is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase and also is a key sensor for oxidative and electrophilic stresses. Here, we first report a novel naturally-occurring mutant of Keap1, designated Keap1ΔC, which lacks most of its C-terminal Nrf2-interacting domain essential for inhibition of the cap’n’collar (CNC) basic-region leucine zipper (bZIP) factor. This mutant Keap1ΔC is yielded by translation from an alternatively mRNA-spliced variant lacking the fourth and fifth exons, but their coding sequences are retained in the wild-type Keap1 locus (with no genomic deletions). Although this variant was found primarily in the human highly-metastatic hepatoma (MHCC97H) cells, it was widely expressed at very lower levels in all other cell lines examined. Such Keap1ΔC retains no or less ability to inhibit Nrf2, so that it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2 due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein

Synergism and Antagonism of Two Distinct, but Confused, Nrf1 Factors in Integral Regulation of the Nuclear-to-Mitochondrial Respiratory and Antioxidant Transcription Networks

There is hitherto no literature available for explaining two distinct, but confused, Nrf1 transcription factors, because they shared the same abbreviations from nuclear factor erythroid 2-related factor 1 (also called Nfe2l1) and nuclear respiratory factor (originally designated α-Pal). Thus, we have here identified that Nfe2l1Nrf1 and α-PalNRF1 exert synergistic and antagonistic roles in integrative regulation of the nuclear-to-mitochondrial respiratory and antioxidant transcription profiles. In mouse embryonic fibroblasts (MEFs), knockout of Nfe2l1-/- leads to substantial decreases in expression levels of α-PalNRF1 and Nfe2l2, together with TFAM (mitochondrial transcription factor A) and other target genes. Similar inhibitory results were determined in Nfe2l2-/- MEFs but with an exception that both GSTa1 and Aldh1a1 were distinguishably upregulated in Nfe2l1-/- MEFs. Such synergistic contributions of Nfe2l1 and Nfe2l2 to the positive regulation of α-PalNRF1 and TFAM were validated in Keap1-/- MEFs. However, human α-PalNRF1 expression was unaltered by hNfe2l1α-/-, hNfe2l2-/-ΔTA, or even hNfe2l1α-/-+siNrf2, albeit TFAM was activated by Nfe2l1 but inhibited by Nfe2l2; such an antagonism occurred in HepG2 cells. Conversely, almost all of mouse Nfe2l1, Nfe2l2, and cotarget genes were downexpressed in α-PalNRF1+/- MEFs. On the contrary, upregulation of human Nfe2l1, Nfe2l2, and relevant reporter genes took place after silencing of α-PalNRF1, but their downregulation occurred upon ectopic expression of α-PalNRF1. Furtherly, Pitx2 (pituitary homeobox 2) was also identified as a direct upstream regulator of Nfe2l1 and TFAM, besides α-PalNRF1. Overall, these across-talks amongst Nfe2l1, Nfe2l2, and α-PalNRF1, along with Pitx2, are integrated from the endoplasmic reticulum towards the nuclear-to-mitochondrial communication for targeting TFAM, in order to finely tune the robust balance of distinct cellular oxidative respiratory and antioxidant gene transcription networks, albeit they differ between the mouse and the human. In addition, it is of crucial importance to note that, in view of such mutual interregulation of these transcription factors, much cautions should be severely taken for us to interpret those relevant experimental results obtained from knockout of Nfe2l1, Nfe2l2, α-Pal or Pitx2, or their gain-of-functional mutants

Method of Estimating Degraded Forest Area: Cases from Dominant Tree Species from Guangdong and Tibet in China

Forest degradation has been considered as one of the main causes of climate change in recent years. The knowledge of estimating degraded forest areas without the application of remote sensing tools can be useful in finding solutions to resolve degradation problems through appropriate restoration methods. Using the existing knowledge through literature review and field-based primary information, we generated new knowledge by combining the information obtained from multi-criteria decision analyses with an analytic hierarchy process, and this was then used to estimate degraded forest area. Estimation involves determining forest degradation index (FDI) and degradation threshold. Continuous inventory data of permanent sample plots collected from degraded forests, consisting of various forest types divided by dominant tree species in the Guangdong province and Tibet autonomous region of China, were used for the purposes. We identified four different forest degradation levels through the determination and comprehensive evaluation of FDI. The degraded forest area with broad-leaved species as dominant tree species in the Guangdong province was estimated to be 83.3% of a total forest area of 24,037 km2. In the same province, the degraded forest area with eucalyptus as a dominant tree species was 59.5% of a total forest area of 18,665 km2. In the Tibet autonomous region, the degraded forest area with spruce as a dominant tree species was 99.1% of a total forest area of 17,614 km2, and with fir as a dominant tree species, the degraded area was 98.4% of a forest area of 12,103 km2. A sampling accuracy of forest areas with national forest inventory was about 95% in both provinces. Our study concludes that the FDI method used has a certain scientific rationality in estimating degraded forest area. The forest provides a variety of tangible and intangible goods and services for humans. Therefore, forest management should focus on the improvement of its overall productivity, which is only possible with improving forest site quality. One of the important steps to improve the quality of a forest site is to resolve its degradation issues. The presented method in this article will be useful in finding the solutions to forest degradation problems. This method, which does not need any remote sensing tool, is simple and can be easily applied for estimating any degraded forest area and developing effective forest restoration plans

Nrf1D Is the First Candidate Secretory Transcription Factor in the Blood Plasma, Its Precursor Existing as a Unique Redox-Sensitive Transmembrane CNC-bZIP Protein in Hemopoietic and Somatic Tissues

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1’s C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood

Nach Is a Novel Subgroup at an Early Evolutionary Stage of the CNC-bZIP Subfamily Transcription Factors from the Marine Bacteria to Humans

Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap’n’collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach (Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans

Both AD2 and NST domains positively regulates chimaeric Gal4-Nrf1 and Gal4-Nrf1β factors.

<p>(<b>A</b>) Schematic representation of expression constructs for Gal4D (Gal4 DNA-binding domain) fusion proteins containing various portions of Nrf1 or Nrf1β (<i>left panel</i>). They were created by ligation of their encoding cDNA fragments into the <i>BamHI/EcoRI</i> sites of the pcDNA3/Gal4-V5 vector. The <i>left panel</i> shows Gal4D-directed reporter activity that was measured from COS-1 cells had been cotransfected with each of indicated expression constructs for the various Gal4D/Nrf1 fusion proteins (1.2 µg), together with <i>P_TKUAS</i>×4<i>-</i>Luc (0.6 µg) and b-gal (0.2 µg) plasmids. The data are shown graphically as fold changes (mean ± S.D.) of transactivation by indicated Gal4-fusion factors when compared with the background (value of 1.0). Significant increases ($, p<0.05 and$$, p<0.001, n = 9) and decreases (*p<0.05, **p<0.001, n = 9) in activity relatively to the referenced activity are indicated (<i>arrows</i>). (<b>B</b>) The above-prepared cell lysates were resolved using 4–12% LDS/NuPAGE and examined by western blotting with V5 antibody. The electrophoretic bands representing free Gal4D and Gal4-Nrf1 fusion proteins are indicated. Samples loaded on each well were calculated to contain equal amounts of β-gal activity.</p

Imaging of fixed and live cells expressing GFP fusion protein with CTD of Nrf1 or its mutants.

<p>(<b>A</b>) Schematic of Six expression constructs for the GFP-CTD fusion protein and its mutants; these fusion proteins have been created by attachment of various lengths of CTD of Nrf1 to the C-terminus of GFP. (<b>B</b>) These indicated expression constructs each were transfected into COS-1 cells for 6 h. The cells were then allowed to recover from transfection in fresh medium for 18 h before being fixed by 4% paraformaldehyde and stained for the nuclear DNA by DAPI. The green signals from GFP were observed under confocal microscope and merged with the DNA-staining images. (<b>C</b> and <b>D</b>) Live-cell imaging of GFP-CTD and its mutant GFP-CTD^Δ731–741(lacking its basic c-tail). COS-1 cells had been transfected with expression constructs for either GFP-CTD (<b><i>C</i></b>) or GFP-CTD^Δ731–741 (<b><i>D</i></b>), together with the ER/DsRed marker, before being subjected to real-time live-cell imaging combined with the <i>in vivo</i> membrane protease protection assay. The cells were permeabilized by digitonin 20 µg/ml) for 10 min, before being co-incubated with PK (50 µg/ml) for 30 min. In the time course, real-time images were acquired using the Leica DMI-6000 microscopy system. The merged images of GFP with ER/DsRed are placed (on <i>the third raw of panels</i>), whereas changes in the intensity of their signals are shown graphically (<i>bottom</i>). Overall, the images shown herein are a representative of at least three independent experiments undertaken on separate occasions that were each performed in triplicate (n = 9). The <i>arrow</i> indicates a ‘hernia-like’ vesicle protruded from the cytoplasm.</p

Opposing regulation of ARE-driven reporter genes by distinct Nrf1 isoforms.

<p>(<b>A</b>) Schematic shows structural domains of five different isoforms of Nrf1. Locations of ER-targeting signal, AD1 and PEST2 are also indicated within distinct domains. (<b>B</b>) Shows luciferase reporter gene activity measured from COS-1 cells that had been co-transfected with 1.2 µg of each expression construct for Nrf1 isoforms, together with 0.6 µg of <i>P_-1061/nqo1</i>-Luc (that is driven by the 1061-bp promoter of <i>Nqo1</i>) and 0.2 µg of β-gal plasmid. The data were calculated as a fold change (mean ± S.D) of transactivation by distinct Nrf1 isoforms. Significant increases ($, p<0.05 and$$, p<0.001, n = 9) and decreases (**, p<0.001, n = 9) in activity were calculated relatively to the background activity (obtained from transfection of cells with an empty pcDNA3 with reporter plasmids). (<b>C</b>) Total lysates of COS-1 cells expressing each of Nrf1 isoforms or Nrf2 were resolved by 12$, p<0.05 and $$, p<0.001, n = 9) and decreases (**, p<0.001, n = 9) in activity relatively to the background activity are indicated. (<b>E</b>) Total lysates of COS-1 cells co-transfected with expression constructs for Nrf1, Nrf1γ and/or Nrf2 alone or in combination (as indicated corresponding to those in panel D) was subject to separation by 4–12% LDS/NuPAGE in a Bis-Tris buffer system. The <i>upper two panels</i> represent similar images from different independent gels, on which location of Nrf2 migration is <i>arrowed</i>, whilst a non-specific protein band is <i>starred</i> (*). The position of the V5-tagged Nrf1 polypeptides of 120, 95, 85, 55, and 36 kDa is indicated. It is notable that the same proteins exhibit distinct mobilities on different electrophoric gels in different running buffer systems (<i>cf. </i><b><i>C</i></b><i> with </i><b><i>E</i></b>).</p