TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

Anti-prion activity of cellulose ether is impaired in mice lacking pre T-cell antigen receptor α, T-cell receptor δ, or lytic granule function.

The anti-prion activity of cellulose ether (CE) has been reported in rodents, but the mechanism of action is not well understood. As defects in early T-cell development have been reported in Tga20 mice which show only a slight effect of CE administration, we investigated the involvement of immune functions in the CE action. We confirmed an insertion of the prion protein transgene into the pre T-cell antigen receptor α gene of Tga20 mice, and its impaired expression in the thymus and other tissues. The influence of immune suppression on the CE effect was then examined in high CE-responder mice treated with immunosuppressive agents or neonatal thymectomy. As neonatal thymectomy significantly reduced the CE effect, we compared the influence of various T-cell defects in mice with similar genetic backgrounds. The CE effect was increased or unchanged in mice with defects in the αß T-cell lineage, whereas it was abolished in T-cell receptor δ deficient mice. Further, when other immune defects were examined, the CE effect was reduced in mice with lysosomal trafficking dysfunction, but was unchanged in mice deficient in B-cell differentiation or toll-like receptor 4 signaling. These findings collectively suggest that the mechanism of CE action may involve γδ T cells and lytic granule function, as well as immune factors like natural killer T cells which are lacking in pre T-cell antigen receptor α deficient mice and neonatally thymectomized mice.

Activities of curcumin-related compounds in two cell lines persistently infected with different prion strains.

BACKGROUND: Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrPSc). In this study, two types of cells persistently infected with prion were treated with curcumin-related compounds. We found that the compounds behave differently in neuroblastoma neuro-2a (N2a) cells infected with different prion strains. METHODS: Curcumin and related compounds were applied to the two types of persistently prion infected cells to analyze the different activities of the compounds. RESULTS: In ScN2a cells, which were infected with the Rocky Mountain Laboratory prion strain, two of the six compounds significantly reduced the PrPSc level in a dose-dependent manner. On the other hand, in N167 cells, effective suppression of the total amount of PrPSc was not observed; instead, two other compounds promoted the formation of covalently linked PrPSc dimers. CONCLUSIONS: Chemometric analysis was used to determine the factors that contributed to the different effects of the six compounds. It showed that the ability to form hydrogen bonds, such as phenolic hydroxyl groups, and hydrophobic molecular properties predominantly contributed to the reduction of the PrPSc level in the ScN2a cells and the dimer formation of PrPSc in the N167 cells, respectively. GENERAL SIGNIFICANCE: The extracted information can be used to delineate the differences among prion strains and to design compounds that are directed toward their respective activities.

Polymorphisms in glia maturation factor ß gene are markers of cellulose ether effectiveness in prion-infected mice.

Anti-prion effects of cellulose ether (CE) are reported in rodents, but the molecular mechanism is fully unknown. Here, we investigated the genetic background of CE effectiveness by proteomic and genetic analysis in mice. Proteomic analysis in the two mouse lines showing a dramatic difference in CE effectiveness revealed a distinct polymorphism in the glia maturation factor ß gene. This polymorphism was significantly associated with the CE effectiveness in various prion-infected mouse lines. Sequencing of this gene and its vicinity genes also revealed several other polymorphisms that were significantly related to the CE effectiveness. These polymorphisms are useful as genetic markers for finding more suitable mouse lines and exploring the genetic factors of CE effectiveness.

Biophysical characterization of heme binding to the intrinsically disordered region of Bach1.

Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.

Functional Heme Binding to the Intrinsically Disordered C-Terminal Region of Bach1, a Transcriptional Repressor.

Heme is one of the key factors involved in the oxidative stress response of cells. The transcriptional repressor Bach1 plays an important role in this response through its heme-binding activity. Heme inhibits the transcriptional-repressor activity of Bach1, and can occur in two binding modes: 5- and 6-coordinated binding. The Cys-Pro (CP) motif has been determined to be the heme-binding motif of Bach family proteins. The sequence of Bach1 includes six CP motifs, and four CP motifs are functional. With the aim of elucidating the molecular mechanism of heme-Bach1 regulation, we conducted biophysical analyses focusing on the C-terminal region of mouse Bach1 (residues 631-739) which is located after the bZip domain and includes one functional CP motif. UV-Vis spectroscopy indicated that the CP motif binds heme via 5-coordinated bond. A mutant, which included a cysteine to alanine substitution at the CP motif, did not show 5-coordination, suggesting that this binding mode is specific to the CP motif. Surface plasmon resonance revealed that the binding affinity and stoichiometry of heme with the Bach1 C-terminal region were KD = 1.37 × 10-5 M and 2.3, respectively. The circular dichroism spectrum in the near-UV region exhibited peaks for heme binding to the CP motif. No significant spectral shifts were observed in the far-UV region when samples with and without heme were compared. Therefore, disordered-ordered transition such as "coupled folding and binding" is not involved in the Bach1-heme system. Consequently, the heme response of this C-terminal region is accomplished by disorder-disorder conformational alteration.

Infection perturbs Bach2- and Bach1-dependent erythroid lineage 'choice' to cause anemia.

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro-myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPß, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPß. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR.

The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.

The double knockout of Bach1 and Bach2 in mice reveals shared compensatory mechanisms in regulating alveolar macrophage function and lung surfactant homeostasis.

Pulmonary alveolar proteinosis (PAP) is a disease resulting from a dysfunction of the alveolar macrophages (AMs) where excess surfactant protein accumulates in the alveolar spaces. We previously reported that Bach2 KO mice developed PAP due to a defect in the handling of lipids by AMs. To investigate the functions of Bach1 and Bach2, which are regulated by oxidative stress, in the AMs and in lung homeostasis, we generated mice that lacked both Bach1 and Bach2 (Bach1/2 DKO mice). The Bach1/2 DKO mice showed more severe PAP phenotype than Bach2 KO mice with abnormal AMs, whereas the Bach1 KO mice did not develop any pulmonary disease. The PAP-like disease in the Bach1/2 DKO and Bach2 KO mice was not ameliorated by antioxidant, suggesting that ROS was not involved in the onset of PAP in the absence of Bach1 and Bach2. A microarray and a chromatin immunoprecipitation sequence analysis revealed that Bach1 and Bach2 directly repress the common set of genes involved in the inflammatory response, and that Bach2 is a major contributor to this repression. These results suggest that Bach1 and Bach2 work in a complementary manner to maintain the normal function of the AMs and surfactant homeostasis in the lung.

Charge-state-distribution analysis of Bach2 intrinsically disordered heme binding region.

Bach2 is a transcriptional repressor that plays an important role in the differentiation of T-cells and B-cells. Bach2 is functionally regulated by heme binding, and possesses five Cys-Pro Cys-Pro (CP)-motifs as the heme binding site. To reveal the molecular mechanism of heme binding by Bach2, the intrinsically disordered heme binding region (a.a. 331-520; Bach2331-520) and its CP-motif mutant were prepared and characterized with and without heme, by UV-Vis spectroscopy and thermal profiles. In addition, the charge-state-distributions (CSDs) were assessed by electrospray ionization mass spectrometry. The UV-Vis spectroscopy revealed a lack of five-coordinated heme binding in the CP-motif mutant of Bach2331-520 The thermal profile and CSDs of Bach2331-520 indicated that heme binding induces the destabilization of Bach2331-520 The thermal profile revealed that the wild type Bach2331-520 was destabilized more than the CP-motif mutant. The shift in the CSDs by heme binding suggested that heme binding causes Bach2331-520 to adopt a more compact conformation. In addition, heme binding to the CP-motif could reduce the flexibility of Bach2331-520 Consequently, the five-coordinated heme binding destabilizes Bach2331-520, by reducing the flexibility of the polypeptide chain.

The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization.

The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells.

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Heme binds to an intrinsically disordered region of Bach2 and alters its conformation.

The transcriptional repressor Bach2 regulates humoral and cellular immunity, including antibody class switching. It possesses a basic leucine zipper domain that mediates DNA binding. Heme inhibits the DNA-binding activity of Bach2 in vitro and induces the degradation of Bach2 in B cells. However, the structural basis of the heme-Bach2 interaction has not been identified. Spectroscopic analyses revealed that Bach2(331-520) is the heme-binding domain, as it includes three Cys-Pro motifs known to be important for heme binding. Heme-titration experiments demonstrated the presence of 5- and 6-coordinated heme-binding modes. Circular dichroism measurements indicated that Bach2(331-520) exists mostly in a random-coil conformation. However, dynamic light scattering analyses showed that, upon heme binding to Bach2(331-520), this region becomes denatured at a lower temperature, as compared with unbound Bach2(331-520). In addition, small-angle X-ray scattering and chemical modification analyses revealed that heme binding induces conformational alterations within the unstructured region. A GAL4-based luciferase assay in 293T cells showed that heme alters the protein interactions mediated by Bach2(331-520). These observations suggested that the unstructured region of Bach2 is important for heme binding, and consequently for its functional regulation.

The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program.

Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2(hi) CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.

Wearing red for signaling: the heme-bach axis in heme metabolism, oxidative stress response and iron immunology.

The connection between gene regulation and metabolism is an old issue that warrants revisiting in order to understand both normal as well as pathogenic processes in higher eukaryotes. Metabolites affect the gene expression by either binding to transcription factors or serving as donors for post-translational modification, such as that involving acetylation and methylation. The focus of this review is heme, a prosthetic group of proteins that includes hemoglobin and cytochromes. Heme has been shown to bind to several transcription factors, including Bach1 and Bach2, in higher eukaryotes. Heme inhibits the transcriptional repressor activity of Bach1, resulting in the derepression of its target genes, such as globin in erythroid cells and heme oxygenase-1 in diverse cell types. Since Bach2 is important for class switch recombination and somatic hypermutation of immunoglobulin genes as well as regulatory and effector T cell differentiation and the macrophage function, the heme-Bach2 axis may regulate the immune response as a signaling cascade. We discuss future issues regarding the topic of the iron/heme-gene regulation network based on current understanding of the heme-Bach axis, including the concept of "iron immunology" as the synthesis of the iron metabolism and the immune response.

Hemopexin-dependent heme uptake via endocytosis regulates the Bach1 transcription repressor and heme oxygenase gene activation.

BACKGROUND: Intracellular heme plays versatile roles in a variety of physiological processes including mitochondrial respiration. Heme also induces the expression of genes such as heme oxygenase-1 (HO-1) by inactivating the transcription repressor Bach1 through direct binding. However, the source of heme for the regulation of the Bach1-HO-1 axis has been unclear. Considering that extracellular heme exists as a complex with hemopexin (Hx) in serum under the physiological conditions, heme-Hx complex may deliver heme for the gene regulation. METHODS: Using a mammalian expression system, high secretory recombinant Hx (rHx) was developed. We examined the effects of rHx-bound heme on HO-1 expression and Bach1 in Hepa-1c1c7 liver cells and THP-1 macrophage cells. We investigated the uptake pathway of rHx-bound heme by treating cells with chlorpromazine (CPZ). RESULTS: rHx-bound heme induced the expression of HO-1 and decreased the level of Bach1 protein. CPZ inhibited the induction of the HO-1 expression by rHx-bound heme. CONCLUSION: rHx-bound heme was internalized into the cells via endocytosis, resulting in HO-1 expression and inactivation of Bach1. GENERAL SIGNIFICANCE: The Bach1-dependent repression of the HO-1 expression is under the control of the Hx-dependent uptake of extracellular heme. Heme may regulate Bach1 as an extracellular signaling molecule.

A sequence-specific DNA glycosylase mediates restriction-modification in Pyrococcus abyssi.

Restriction-modification systems consist of genes that encode a restriction enzyme and a cognate methyltransferase. Thus far, it was believed that restriction enzymes are sequence-specific endonucleases that introduce double-strand breaks at specific sites by catalysing the cleavages of phosphodiester bonds. Here we report that based on the crystal structure and enzymatic activity, one of the restriction enzymes, R.PabI, is not an endonuclease but a sequence-specific adenine DNA glycosylase. The structure of the R.PabI-DNA complex shows that R.PabI unwinds DNA at a 5'-GTAC-3' site and flips the guanine and adenine bases out of the DNA helix to recognize the sequence. R.PabI catalyses the hydrolysis of the N-glycosidic bond between the adenine base and the sugar in the DNA and produces two opposing apurinic/apyrimidinic (AP) sites. The opposing AP sites are cleaved by heat-promoted ß elimination and/or by endogenous AP endonucleases of host cells to introduce a double-strand break.

Heme regulates B-cell differentiation, antibody class switch, and heme oxygenase-1 expression in B cells as a ligand of Bach2.

Heme binds to proteins to modulate their function, thereby functioning as a signaling molecule in a variety of biologic events. We found that heme bound to Bach2, a transcription factor essential for humoral immunity, including antibody class switch. Heme inhibited the DNA binding activity of Bach2 in vitro and reduced its half-life in B cells. When added to B-cell primary cultures, heme enhanced the transcription of Blimp-1, the master regulator of plasma cells, and skewed plasma cell differentiation toward the IgM isotype, decreasing the IgG levels in vitro. Intraperitoneal injection of heme in mice inhibited the production of antigen-specific IgM when heme was administered simultaneously with the antigen but not when it was administered after antigen exposure, suggesting that heme also modulates the early phase of B-cell responses to antigen. Heme oxygenase-1, which is known to be regulated by heme, was repressed by both Bach2 and Bach1 in B cells. Furthermore, the expression of genes for heme uptake changed in response to B-cell activation and heme administration. Our results reveal a new function for heme as a ligand of Bach2 and as a modulatory signal involved in plasma cell differentiation.

Crystal structure of the Bach1 BTB domain and its regulation of homodimerization.

The BTB/POZ domain is known as a protein-protein interaction motif that mediates homodimer and higher order self-associations. Proteins containing the BTB domain exist throughout eukaryotes; however, there is little information about the mechanism that determines the oligomeric state of the BTB domain. To address this question, we have determined the X-ray structure of the mouse Bach1 BTB domain. The present structure is similar to the previously determined BTB domain folds, including the human Bach1 BTB domain; however, distinct structural features are present, such as a novel homodimer interaction surface. The homodimer formation was found to involve a novel hydrogen bond network and interactions between hydrophobic surfaces of the kinked N-terminus (N-hook) and the partner's C-terminal residues. The deletion of the N-hook resulted in the conversion of the homodimer into a monomer in solution, indicating that the N-hook promotes the homodimerization of the mBach1 BTB domain. We have also found that the BTB domain of Bach2, a protein highly related to Bach1, is present as a monomer due to a short peptide insertion at the N-hook. These results represent the first example of the key modulatory element of BTB domain homodimerization.

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity.

Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.