Yuancai Xiang

The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors.

The C-terminal domain of Nrf1 negatively regulates the full-length CNC-bZIP factor and its shorter isoform LCR-F1/Nrf1β; both are also inhibited by the small dominant-negative Nrf1γ/δ isoforms that down-regulate ARE-battery gene expression.

The C-terminal domain (CTD, aa 686–741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1β (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ.

TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells

The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α−/− cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α−/− cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α−/− cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration).

A naturally-occurring dominant-negative competitor of Keap1 against its inhibition of Nrf2

Transcription factor Nrf2 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) as an adaptor subunit of Cullin 3-based E3 ubiquitin ligase, that is a key sensor for oxidative and electrophilic stresses. Now, 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 CNC-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. No matter whether Keap1ΔC retains less or no ability to inhibit Nrf2, it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2-target genes. This is due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein.

Nrf1D is the first candidate secretory transcription factor in the blood plasma, with its precursor existing as a unique redox-sensitive transmembrane CNC-bZIP protein in somatic tissues

Amongst multiple distinct isoforms, Nrf1D is synthesized from translation of an alternatively-spliced transcript of Nrf1 mRNA, with a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the 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 identified to fold into a redox-sensitive transmembrane domain that enables it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D confers it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with only a less ability than wild-type Nrf1 at mediating target gene expression. Further evidence has been presented revealing that both mRNA and protein levels of Nrf1D were detected to varying extents in somatic tissues. Surprisingly, we also found the existence of Nrf1D-derived isoforms in the blood plasma, implying that it is a candidate secretory transcription factor, although its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies, before being unleashed from the ER to enter the blood plasma.

Nach is a novel ancestral subfamily of the CNC-bZIP transcription factors selected during evolution from the marine bacteria to human

All living organisms have undergone the evolutionary selection under the changing natural environments to survive as diverse life forms. All life processes including normal homeostatic development and growth into organismic bodies with distinct cellular identifications, as well as their adaptive responses to various intracellular and environmental stresses, are tightly controlled by signaling of transcriptional networks towards regulation of cognate genes by many different transcription factors. Amongst them, one of the most conserved is the basic-region leucine zipper (bZIP) family. They play vital roles essential for cell proliferation, differentiation and maintenance in complex multicellular organisms. Notably, an unresolved divergence on the evolution of bZIP proteins is addressed here. By a combination of bioinformatics with genomics and molecular biology, we have demonstrated that two of the most ancestral family members classified into BATF and Jun subgroups are originated from viruses, albeit expansion and diversification of the bZIP superfamily occur in different vertebrates. Interestingly, a specific ancestral subfamily of bZIP proteins is identified and also designated Nach (Nrf and CNC homology) on account of their highly conservativity with NF-E2 p45 subunit-related factors Nrf1/2. Further experimental evidence reveals that Nach1/2 from the marine bacteria exerts distinctive functions from Nrf1/2 in the transcriptional ability to regulate antioxidant response element (ARE)-driven cytoprotective genes. Collectively, an insight into Nach/CNC-bZIP proteins provides a better understanding of distinct biological functions between these factors selected during evolution from the marine bacteria to human. Significance We identified the novel ancestral subfamily (i.e. Nach) of CNC-bZIP transcription factors with highly conservativity from marine bacteria to human. Combination of bioinformatics with genomics and molecular biology demonstrated that two of the most ancestral family members classified into BATF and Jun subgroups are originated from viruses. The Jun and CNC subfamilies also share a common origin of these bZIP proteins. Further experimental evidence reveals that Nach1/2 from the marine bacteria exerts nuance functions from human Nrf1/2 in the transcriptional ability to regulate antioxidant response element (ARE)-driven genes, responsible for the host cytoprotection against inflammation and cancer. Overall, this study is of multidisciplinary interests to provide a better understanding of distinct biological functions between Nach/CNC-bZIPs selected during evolution.

Mechanisms controlling the multistage post-translational processing of endogenous Nrf1α/TCF11 proteins to yield distinct isoforms within the coupled positive and negative feedback circuits

&NA; To gain a better understanding of the multistep processing of Nrf1 to yield various isoforms with confused molecular masses, we herein establish a generally acceptable criterion required for identification of its endogenous full‐length proteins and derivative isoforms expressed differentially in distinct experimental cell lines. Further work has been focused on the molecular mechanisms that dictate the successive post‐translational modifications (i.e. glycosylation by OST, deglycosylation by NGLY, and ubiquitination by Hrd1) of this CNC‐bZIP protein and its proteolytic processing to give rise to multiple proteoforms. Several lines of experimental evidence have demonstrated that the nascent Nrf1&agr;/TCF11 polypeptide (non‐glycosylated) is transiently translocated into the endoplasmic reticulum (ER), in which it becomes an inactive glycoprotein‐A, and is folded in a proper topology within and around membranes. Thereafter, dynamic repositioning of the ER‐resident domains in Nrf1 glycoprotein is driven by p97‐fueled retrotranslocation into extra‐ER compartments. Therein, Nrf1 glycoprotein is allowed for deglycosylation digestion by glycosidases into a deglycoprotein‐B and its progressive proteolytic processing by cytosolic DDI‐1/2 and proteasomes so as to generate N‐terminally‐truncated protein‐C/D. This processing is accompanied by removal of a major N‐terminal ˜12.5‐kDa polypeptide from Nrf1&agr;. Interestingly, our present study has further unraveled that there exist coupled positive and negative feedback circuits between Nrf1 and cognate target genes, including those encoding its regulators p97, Hrd1, DDI‐1 and proteasomes. These key players are differentially or even oppositely involved in diverse cellular signaling responses to distinct extents of ER‐derived proteotoxic and oxidative stresses induced by different concentrations of proteasomal inhibitors. Graphical abstract Figure. No caption available. HighlightsThe possessing of endogenous Nrf1&agr;/TCF11 yields four major isoforms A, B, C and D.Distinct Nrf1&agr;/TCF11‐derived isoforms of between ˜140‐ and 75‐kDa are identified.Mechanisms for the Nrf1 post‐synthetic modification and processing are elucidated.Regulation of Nrf1 by target genes occurs in positive and negative feedback circuits.

Topovectorial mechanisms control the juxtamembrane proteolytic processing of Nrf1 to remove its N-terminal polypeptides during maturation of the CNC-bZIP factor

&NA; The topobiological behaviour of Nrf1 dictates its post‐translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of Nrf1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N‐terminal domain (NTD, also refolded into a UBL module) and acidic domain‐1 (AD1) to yield multiple isoforms. Notably, an N‐terminal ˜12.5‐kDa polypeptide of Nrf1 arises from selective cleavage at an NHB2‐adjoining region within NTD, whilst other longer UBL‐containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of Nrf1 to proteolysis is determined by dynamic repositioning of potential UBL‐adjacent degrons and cleavage sites from the ER lumen through p97‐driven retrotranslocation and ‐independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of Nrf1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI‐1/2 proteases and also partiality degraded via 26S proteasomes. The resultant proteolytic processing of Nrf1 gives rise to a mature ˜85‐kDa CNC‐bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of Nrf1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of Nrf1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1. Graphical abstract Figure. No caption available. HighlightsThe multistep processing of Nrf1 is dictated by its topovectorial mechanisms.The regulated juxtamembrane proteolysis of Nrf1 occurs in close proximity to the ER.Removal of distinct N‐terminal peptides from Nrf1 gives rise to a mature isoform.Cytosolic proteases are required for the selective juxtamembrane proteolysis of Nrf1.

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.

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.