Crystal structure of heme A synthase from Bacillus subtilis.

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from Bacillus subtilis at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in B. subtilis, is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis.

Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å.

The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αß-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.

Transcriptional repression of HER2 by ANO1 Cl- channel inhibition in human breast cancer cells with resistance to trastuzumab.

The Ca2+-activated Cl- channel ANO1 contributes to tumorigenesis and metastasis in several carcinomas including breast cancer (BCA). Cl- channels have recently been attracting attention as 'transcriptional modulators'. Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 30% of patients with BCA, and anti-HER2 monoclonal antibodies such as trastuzumab have emerged as a treatment for metastatic BCA. Among the seven human BCA cell lines examined in the present study, MDA-MB-453 and YMB-1 cells were HER2-positive; however, YMB-1 cell viability showed resistance to trastuzumab. Whole-cell patch-clamp configurations indicated that ANO1 was the main Cl- conductance in YMB-1 cells, and the pharmacological and siRNA-mediated inhibition of ANO1 significantly prevented HER2 transcription in YMB-1 cells. The expression levels of insulin-like growth factor-binding protein 5 (IGFBP5), which is a risk factor for BCA recurrence and metastasis, was not affected by the inhibition of ANO1 in YMB-1 cells. These results suggest that ANO1 Cl- channels may function as a transcriptional regulator of HER2, and ANO1 inhibitors have potential in the treatment of BCA patients with resistance to HER2-targeted therapy.

Defective splicing of the background K+ channel K2P5.1 by the pre-mRNA splicing inhibitor, pladienolide B in lectin-activated mouse splenic CD4+ T cells.

The two-pore domain K+ channel K2P5.1 has been implicated in the pathogenesis of autoimmune diseases. We investigated the changes in K2P5.1 activity caused by a defect in normal pre-mRNA splicing in concanavalin A-activated mouse splenic CD4+ T cells. The pre-mRNA splicing inhibitor, pladienolide B (1 µM) markedly decreased full-length K2P5.1 transcription in activated CD4+ T cells, resulting in the disappearance of K2P5.1 activity and an imbalance in Th17 and Treg cytokines. These results suggest that the defect in K2P5.1 splicing by the pre-mRNA splicing inhibitor regulates pro- and/or anti-inflammatory cytokine production in K2P5.1-associated autoimmune diseases.

Down-Regulation of Ca2+-Activated K⁺ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists.

Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K⁺ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

Downregulation of Ca2+-activated Cl- channel TMEM16A by the inhibition of histone deacetylase in TMEM16A-expressing cancer cells.

The Ca(2+)-activated Cl(-) channel transmembrane proteins with unknown function 16 A (TMEM16A; also known as anoctamin 1 or discovered on gastrointestinal stromal tumor 1) plays an important role in facilitating the cell growth and metastasis of TMEM16A-expressing cancer cells. Histone deacetylase (HDAC) inhibitors (HDACi) are useful agents for cancer therapy, but it remains unclear whether ion channels are epigenetically regulated by them. Using real-time polymerase chain reaction, Western blot analysis, and whole-cell patch-clamp assays, we found a significant decrease in TMEM16A expression and its functional activity was induced by the vorinostat, a pan-HDACi in TMEM16A-expressing human cancer cell lines, the prostatic cancer cell line PC-3, and the breast cancer cell line YMB-1. TMEM16A downregulation was not induced by the chemotherapy drug paclitaxel in either cell type. Pharmacologic blockade of HDAC3 by 1 µM T247 [N-(2-aminophenyl)-4-[1-(2-thiophen-3-ylethyl)-1H-[1],[2],[3]triazol-4-yl]benzamide], a HDAC3-selective HDACi, elicited a large decrease in TMEM16A expression and functional activity in both cell types, and pharmacologic blockade of HDAC2 by AATB [4-(acetylamino)-N-[2-amino-5-(2-thienyl)phenyl]-benzamide; 300 nM] elicited partial inhibition of TMEM16A expression (∼40%) in both. Pharmacologic blockade of HDAC1 or HDAC6 did not elicit any significant change in TMEM16A expression, respectively. In addition, inhibition of HDAC3 induced by small interfering RNA elicited a large decrease in TMEM16A transcripts in both cell types. Taken together, in malignancies with a frequent gene amplification of TMEM16A, HDAC3 inhibition may exert suppressive effects on cancer cell viability via downregulation of TMEM16A.

Detailed analysis of distorted retinal and its interaction with surrounding residues in the K intermediate of bacteriorhodopsin.

The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.

Structure of a putative immature form of a Rieske-type iron-sulfur protein in complex with zinc chloride.

Iron-sulfur clusters are prosthetic groups of proteins involved in various biological processes. However, details of the immature state of the iron-sulfur cluster into proteins have not yet been elucidated. We report here the first structural analysis of the Zn-containing form of a Rieske-type iron-sulfur protein, PetA, from Thermochromatium tepidum (TtPetA) by X-ray crystallography and small-angle X-ray scattering analysis. The Zn-containing form of TtPetA was indicated to be a dimer in solution. The zinc ion adopts a regular tetra-coordination with two chloride ions and two cysteine residues. Only a histidine residue in the cluster-binding site exhibited a conformational difference from the [2Fe-2S] containing form. The Zn-containing structure indicates that the conformation of the cluster binding site is already constructed and stabilized before insertion of [2Fe-2S]. The binding mode of ZnCl2, similar to the [2Fe-2S] cluster, suggests that the zinc ions might be involved in the insertion of the [2Fe-2S] cluster.

Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, K(Ca)3.1, in immune function of lymphoid cells.

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Down-regulation of the large-conductance Ca(2+)-activated K+ channel, K(Ca)1.1 in the prostatic stromal cells of benign prostate hyperplasia.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel encoded by K(Ca)1.1 plays an important role in the control of smooth muscle tone by modulating membrane potential and intracellular Ca(2+) mobilization. BK(Ca) channel is functionally expressed in prostatic smooth muscle cells, and is activated by α(1)-adrenoceptor agonists. The main objective of this study was to elucidate the pathophysiological significance of changes in prostatic K(Ca)1.1 expressions in benign prostatic hyperplasia (BPH). Our previous study has shown that K(Ca)3.1 encoding intermediate-conductance K(Ca) (IK(Ca)) channel is up-regulated in stromal cells of implanted urogenital sinuses (UGSs) of stromal hyperplasia BPH model rats and in those of prostatic tissues from BPH patients. In the present study, the results from real-time polymerase chain reaction (PCR), Western blot, and immunohistochemical analyses showed significant down-regulation of K(Ca)1.1 transcripts and proteins and negative correlation between K(Ca)1.1 and K(Ca)3.1 transcript expressions in prostatic stromal cells of both BPH model rats and BPH patients. Corresponding to down-regulation of K(Ca)1.1 expression in stromal cells of implanted UGSs, membrane depolarization by application of the BK(Ca) channel blocker was disappeared. Down-regulation of K(Ca)1.1 may be involved in the phenotype switch from contractile profile to proliferative one in prostatic stromal cells of BPH patients.

Intermediate-conductance Ca2+-activated K+ channel, KCa3.1, as a novel therapeutic target for benign prostatic hyperplasia.

Recently, a new experimental stromal hyperplasia animal model corresponding to clinical benign prostatic hyperplasia (BPH) was established. The main objective of this study was to elucidate the roles of the intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) in the implanted urogenital sinus (UGS) of stromal hyperplasia BPH model rats. Using DNA microarray, real-time polymerase chain reaction, Western blot, and/or immunohistochemical analyses, we identified the expression of K(Ca)3.1 and its transcriptional regulators in implanted UGS of BPH model rats and prostate needle-biopsy samples and surgical prostate specimens of BPH patients. We also examined the in vivo effects of a K(Ca)3.1 blocker, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), on the proliferation index of implanted UGS by measurement of UGS weights and proliferating cell nuclear antigen immunostaining. K(Ca)3.1 genes and proteins were highly expressed in implanted UGS rather than in the normal host prostate. In the implanted UGS, the gene expressions of two transcriptional regulators of K(Ca)3.1, repressor element 1-silencing transcription factor and c-Jun, were significantly down- and up-regulated, and the regulations were correlated negatively or positively with K(Ca)3.1 expression, respectively. Positive signals of K(Ca)3.1 proteins were detected exclusively in stromal cells, whereas they were scarcely immunolocalized to basal cells of the epithelium in implanted UGS. In vivo treatment with TRAM-34 significantly suppressed the increase in implanted UGS weights compared with the decrease in stromal cell components. Moreover, significant levels of K(Ca)3.1 expression were observed in human BPH samples. K(Ca)3.1 blockers may be a novel treatment option for patients suffering from BPH.

[Construction and evaluation of new anti-cancer drug prescription support system to promote proper use of anticancer drugs].

In the Nagoya City University Hospital, we started an Anti-cancer drug prescription support system according to which physicians input the injection prescription order from the regimen, together with the Outpatient Oncology Unit established in May, 2007. In order to prepare the anti-cancer drug more safely, we constructed a new Anti-cancer drug preparation support system(new system) at the same time. We investigated and evaluated the time and accuracy required for the preparation between the old and new systems. In the old system, we used electronic calculators or manual methods to perform calculations in the prescription procedure. In the new system, notes are automatically printed out with the kind, amount, and extraction amount of the dissolution liquid according to the dosage of the given anti-cancer drug for the injection prescription. Therefore, even a person with little experience in the preparation can confirm the preparative procedure accurately and promptly. Moreover, this system improves the efficiency of the preparation and it is thought that the utility is high as a part of the risk management.

Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells.

The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory ß and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.

Crystallographic characterization of the high-potential iron-sulfur protein in the oxidized state at 0.8 Å resolution.

High-potential iron-sulfur protein (HiPIP) is a soluble electron carrier protein of photosynthetic bacteria with an Fe4S4 cluster. Although structural changes accompanying the electron transfer are important for understanding of the functional mechanism, the changes have not been clarified in sufficient detail. We previously reported the high-resolution crystal structures of HiPIP from a thermophilic purple bacterium Thermochromatium tepidum in the reduced state. In order to perform a detailed comparison between the structures in different redox states, the oxidized structure should also be revealed at high resolution. Therefore, in the present study we performed a crystallographic analysis of oxidized HiPIP and a structural comparison with the reduced form at a high resolution of 0.8 Å. The comparison highlighted small but significant contraction in the iron-sulfur cluster. The changes in Fe-S bond lengths were similar to that predicted by theoretical calculation, although some discrepancies were also found. Almost distances between the sulfur atoms of the iron-sulfur cluster and the protein environment are elongated upon the oxidation. Positional changes of hydrogen atoms in the protein environment, such as on the amide-hydrogen of Cys75 in the proximity of the iron-sulfur cluster, were also observed in the accurate analyses. None of the water molecules exhibited significant changes in position or anisotropy of atomic displacement parameter between the two states, while the orientations of some water molecules were different.

Crystal structures of chitin binding domains of chitinase from Thermococcus kodakarensis KOD1.

Chitinase from T. kodakarensis (TkChiA) catalyzes the hydrolysis of chitin. The enzyme consists of two catalytic and three binding domains (ChBD1, ChBD2 and ChBD3). ChBD2 and ChBD3 can bind to not only chitin but also cellulose. In both domains, the intervals of the side chains of the three tryptophan residues, which are located on the molecular surface, correspond to twice the length of the lattice of the chitin. A binding model with crystalline chitin implies that the tryptophan residues and a glutamate residue interact with the hexose ring by CH-π interactions and the amide group by a hydrogen bond, respectively.

Downregulation of the Ca(2+)-activated K(+) channel KC a3.1 by histone deacetylase inhibition in human breast cancer cells.

The intermediate-conductance Ca(2+)-activated K(+) channel KC a3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target and biomarker for cancer. Histone deacetylase inhibitors (HDACis) have considerable potential for cancer therapy, however, the effects of HDACis on ion channel expression have not yet been investigated in detail. The results of this study showed a significant decrease in KC a3.1 transcription by HDAC inhibition in the human breast cancer cell line YMB-1, which functionally expresses KCa3.1. A treatment with the clinically available, class I, II, and IV HDAC inhibitor, vorinostat significantly downregulated KC a3.1 transcription in a concentration-dependent manner, and the plasmalemmal expression of the KC a3.1 protein and its functional activity were correspondingly decreased. Pharmacological and siRNA-based HDAC inhibition both revealed the involvement of HDAC2 and HDAC3 in KC a3.1 transcription through the same mechanism. The downregulation of KC a3.1 in YMB-1 was not due to the upregulation of the repressor element-1 silencing transcription factor, REST and the insulin-like growth factor-binding protein 5, IGFBP5. The significant decrease in KC a3.1 transcription by HDAC inhibition was also observed in the KC a3.1-expressing human prostate cancer cell line, PC-3. These results suggest that vorinostat and the selective HDACis for HDAC2 and/or HDAC3 are effective drug candidates for KC a3.1-overexpressing cancers.

Malignancy grade-dependent expression of K+-channel subtypes in human prostate cancer.

K(+) channels are key molecules in the progression of several cancer types and considered to be potential targets for cancer therapy. We examined the gene expressions of voltage-gated (K(v)), Ca(2+)-activated (K(Ca)), and two-pore domain (K(2P)) K(+)-channel subtypes in needle-biopsy samples of human prostate cancer (PCa) by real-time PCR and compared them with those in PCa epithelial cell lines. The expression of K(v)1.3, K(Ca)1.1, K(Ca)3.1, and K(2P)1 markedly increased in the PCa group with Gleason score of 5 - 6 (GS5-6) but significantly decreased in the GS8-9 group. This malignancy grade-dependent K(+)-channel expression pattern may provide a convenient marker to understand PCa progression level.