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.

Down-Regulation of CYP3A4 by the KCa1.1 Inhibition Is Responsible for Overcoming Resistance to Doxorubicin in Cancer Spheroid Models.

The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting KCa1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of KCa1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting KCa1.1 in cancer spheroid models, and KCa1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.

Empagliflozin induces the transcriptional program for nutrient homeostasis in skeletal muscle in normal mice.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve heart failure (HF) outcomes across a range of patient characteristics. A hypothesis that SGLT2i induce metabolic change similar to fasting has recently been proposed to explain their profound clinical benefits. However, it remains unclear whether SGLT2i primarily induce this change in physiological settings. Here, we demonstrate that empagliflozin administration under ad libitum feeding did not cause weight loss but did increase transcripts of the key nutrient sensors, AMP-activated protein kinase and nicotinamide phosphoribosyltransferase, and the master regulator of mitochondrial gene expression, PGC-1α, in quadriceps muscle in healthy mice. Expression of these genes correlated with that of PPARα and PPARδ target genes related to mitochondrial metabolism and oxidative stress response, and also correlated with serum ketone body ß-hydroxybutyrate. These results were not observed in the heart. Collectively, this study revealed that empagliflozin activates transcriptional programs critical for sensing and adaptation to nutrient availability intrinsic to skeletal muscle rather than the heart even in normocaloric condition. As activation of PGC-1α is sufficient for metabolic switch from fatigable, glycolytic metabolism toward fatigue-resistant, oxidative mechanism in skeletal muscle myofibers, our findings may partly explain the improvement of exercise tolerance in patients with HF receiving empagliflozin.

Pathophysiologic Contributions of Visceral Adiposity to Left Ventricular Diastolic Dysfunction.

BACKGROUND: Visceral fat produces inflammatory cytokines and may play a major role in heart failure with preserved ejection fraction (HFpEF). However, little data exist regarding how qualitative and quantitative abnormalities of visceral fat would contribute to left ventricular diastolic dysfunction (LVDD). METHODS: We studied 77 participants who underwent open abdominal surgery for intra-abdominal tumors (LVDD, n = 44; controls without LVDD, n = 33). Visceral fat samples were obtained during the surgery, and mRNA levels of inflammatory cytokines were measured. Visceral and subcutaneous fat areas were measured using abdominal computed tomography. RESULTS: Patients with significant LVDD had greater LV remodeling and worse LVDD than controls. While body weight, body mass index, and subcutaneous fat area were similar in patients with LVDD and controls, the visceral fat area was larger in patients with LVDD than in controls. The visceral fat area was correlated with BNP levels, LV mass index, mitral e' velocity, and E/e' ratio. There were no significant differences in the mRNA expressions of visceral adipose tissue cytokines (IL-2, -6, -8, and -1ß, TNFα, CRP, TGFß, IFNγ, leptin, and adiponectin) between the groups. CONCLUSIONS: Our data may suggest the pathophysiological contribution of visceral adiposity to LVDD.

Effect of nasal mupirocin treatment on extranasal carriage of methicillin-resistant Staphylococcus aureus among pediatric patients admitted to the neonatal intensive care unit.

Decolonization of MRSA detected in the oral cavity and tracheal aspirates occurred in 85% and 58% of neonates, respectively, with nasal mupirocin treatment. Recurrent MRSA colonization occurred in 45% of neonates whose MRSA was detected in the oral cavity at a mean of 19 days. Recurrent MRSA colonization occurred in 58% of neonates whose MRSA was detected in tracheal aspirates at a mean of 23 days.

Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages.

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.

A Simple Flow Injection Analysis-Tandem Mass Spectrometry Method to Reduce False Positives of C5-Acylcarnitines Due to Pivaloylcarnitine Using Reference Ions.

Flow injection analysis−tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

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.

KCa3.1 inhibition-induced activation of the JNK/c-Jun signaling pathway enhances IL-10 expression in peripherally-induced regulatory T cells.

The KCa3.1 inhibition up-regulates IL-10 expression in regulatory T (Treg) cells in the recovery phase of inflammatory bowel disease (IBD) model mice; however, the underlying signaling pathway remains unclear. We investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its transcription factors (TFs) in in vitro-induced mouse splenic Treg cells. The pharmacological inhibition of JNK reversed KCa3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of KCa3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the KCa3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced Treg cells.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model.

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons.

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin-biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions.

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.

Increased Interleukin-10 Expression by the Inhibition of Ca2+-Activated K+ Channel KCa3.1 in CD4+CD25+ Regulatory T Cells in the Recovery Phase in an Inflammatory Bowel Disease Mouse Model.

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract arising from abnormal responses of the innate and adaptative immune systems. Interleukin (IL)-10-producing CD4+CD25+ regulatory T (Treg) cells play a protective role in the recovery phase of IBD. In the present study, the effects of the administration of the selective Ca2+-activated K+ channel KCa3.1 inhibitor TRAM-34 on disease activities were examined in chemically induced IBD model mice. IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage in the colon, was significantly lower in mice administered 1 mg/kg TRAM-34 than in vehicle-administered mice. Quantitative real-time polymerase chain reaction examinations showed that IL-10 expression levels in the recovery phase were markedly increased by the inhibition of KCa3.1 in mesenteric lymph node (mLN) Treg cells of IBD model mice compared with vehicle-administered mice. Among several positive and negative transcriptional regulators (TRs) for IL-10, three positive TRs-E4BP4, KLF4, and Blimp1-were upregulated by the inhibition of KCa3.1 in the mLN Treg cells of IBD model mice. In mouse peripheral CD4+CD25+ Treg cells induced by lectin stimulation, IL-10 expression and secretion were enhanced by the treatment with TRAM-34, together with the upregulation of E4BP4, KLF4, and Blimp1. Collectively, the present results demonstrated that the pharmacological inhibition of KCa3.1 decreased IBD symptoms in the IBD model by increasing IL-10 production in peripheral Treg cells and that IL-10high Treg cells produced by the treatment with KCa3.1 inhibitor may contribute to efficient Treg therapy for chronic inflammatory disorders, including IBD. SIGNIFICANCE STATEMENT: Pharmacological inhibition of Ca2+-activated K+ channel KCa3.1 increased IL-10 expression in peripheral Treg cells, together with the upregulation of the transcriptional regulators of IL-10: Krüppel-like factor 4, E4 promoter-binding protein 4, and/or B lymphocyte-induced maturation protein 1. The manipulation of IL-10high-producing Treg cells by the pharmacological inhibition of KCa3.1 may be beneficial in the treatment of chronic inflammatory diseases such as inflammatory bowel disease.

Reversibility of Rocuronium-Induced Deep Neuromuscular Block with Sugammadex in Infants and Children-A Randomized Study.

Sugammadex 4 mg·kg-1 is recommended for reversal from rocuronium-induced deep neuromuscular block. However, there is limited data regarding the dose-response of sugammadex required for reversal from deep neuromuscular block in pediatric patients. The aim of this study was to determine the reversibility of rocuronium-induced deep neuromuscular block with sugammadex in infants and children. Seventy-five children (48 infants and 27 children, mean standard deviation (S.D.), age: 11.6 (6.7) months) were enrolled in this study. After induction of anesthesia and administration of 0.6 mg·kg-1 rocuronium, neuromuscular block was acceleromyographically evaluated by observing contractions of the adductor pollicis muscle to ulnar nerve train-of-four (TOF) stimulation. Subsequently, the intensity of rocuronium-induced block was determined every 6 min using post-tetanic count (PTC) stimulation during sevoflurane and remifentanil anesthesia. When the first response to the PTC stimulus was detected, either 1, 2 or 4 mg·kg-1 sugammadex was administered and the time required for facilitated recovery to a TOF ratio of 0.9 following each dose was compared. The time [mean (S.D.)] from the administration of 1 mg·kg-1 sugammadex until recovery to a TOF ratio of 0.9 was significantly longer [129.1 (83.5) s, p < 0.001] than that with 2 and 4 mg·kg-1 sugammadex [70.3 (26.7) s and 68.2 (34.5) s, respectively]. Incomplete reversal was seen in 3 patients in the 1 mg·kg-1 group. The results suggested that a 4 mg·kg-1 sugammadex dose is recommended for reversal from rocuronium-induced deep neuromuscular block even in infants and children.

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.