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Ryu Nishimura,
Tomokazu Shibata,
Hulin Tai,
Izumi Ishigami,
Takashi Ogura,
Satoshi Nagao,
Takashi Matsuo,
Shun Hirota,
Kiyohiro Imai,
Saburo Neya,
Akihiro Suzuki, Yasuhiko Yamamoto
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ABSTRACT: We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (νCO) increases with decreasing electron density of the heme Fe atom (ρFe). This finding demonstrated that the νCO value can be used as a sensitive measure of the ρFe value and that the π back-donation of the heme Fe atom to CO is affected by the heme π-system perturbation induced through peripheral side chain modifications.
Inorganic Chemistry 02/2013; · 4.60 Impact Factor
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ABSTRACT: The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low-molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an anti-tumor effect beyond the known activity of anticoagulation. However, the link between the anti-RAGE and anti-tumor activities of LMWH has not yet to be fully elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion and metastasis via blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full-length RAGE-overexpressing (HT1080(RAGE) ), RAGE dominant-negative, intracellular tail-deleted RAGE-overexpressing (HT1080(dn) (RAGE) ) and mock-transfected control (HT1080(mock) ) cells. Confocal microscopy demonstrated the expression of HMGB1 and RAGE in HT1080 cells. LMWH significantly inhibited HMGB1-induced NFκB activation via RAGE using an NFκB-dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE-expression attenuated, HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Administration of LMWH significantly inhibited the migration, invasion, tumor formation and lung metastasis of HT1080(RAGE) cells, but not of HT1080(mock) or HT1080(dn) (RAGE) cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. LMWH is thus suggested to have therapeutic potential in patients with certain types of malignant tumors.
Cancer Science 02/2013; · 3.33 Impact Factor
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ABSTRACT: Glucolipotoxicity, which is exerted by free fatty acids (FFA) and prolonged hyperglycemia, is implicated in pancreatic β-cell failure in diabetes. Pattern recognition receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptors 2 and 4 could mediate danger signals in β-cells. We examined whether RAGE contributes to β-cell failure in a type 2 diabetes mouse model. Pancreatic islets were isolated from ob/ob, db/db, diet-induced obesity (DIO), RAGE-null (RAGE(-/-) ), and RAGE(+/+) wild-type (WT) control mice and dispersed into single cells for flow cytometry. RAGE expression was detected in insulin-positive β-cells of ob/ob and db/db mice, but not of WT, DIO, or RAGE(-/-) mice: thus, inadequate leptin receptor signaling and RAGE expression may be linked. Compared with RAGE(+/+) db/db mice, RAGE(-/-) db/db mice showed higher β-cell number and mass with less apoptosis as well as glucose tolerance with higher insulin secretion without any differences in serum levels of FFA and adiponectin. Palmitate or oleate pretreatment combined with a leptin antagonist induced RAGE expression, AGE-elicited apoptosis, and impaired glucose-stimulated insulin secretion by advanced glycation end products (AGE) in MIN6 cells. FFA elevation with concomitant AGE formation during prolonged hyperglycemia could cause β-cell damage through insufficient leptin action and subsequent RAGE induction in type 2 diabetes.
Genes to Cells 02/2013; · 2.68 Impact Factor
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Tomokazu Shibata,
Daichi Matsumoto,
Ryu Nishimura,
Hulin Tai,
Ariki Matsuoka,
Satoshi Nagao,
Takashi Matsuo,
Shun Hirota,
Kiyohiro Imai,
Saburo Neya,
Akihiro Suzuki, Yasuhiko Yamamoto
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ABSTRACT: Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe(3+)-O(2)(-)-like species has been expected for the Fe(2+)-O(2) bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe(2+)-O(2) form results in lowering of the O(2) affinity due to an increase in the O(2) dissociation rate. On the other hand, a shift of the resonance toward the Fe(3+)-O(2)(-)-like species results in acceleration of the autoxidation through increasing H(+) affinity of the bound ligand.
Inorganic Chemistry 10/2012; · 4.60 Impact Factor
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Masayo Monden,
Hidenori Koyama,
Yoshiko Otsuka,
Tomoaki Morioka,
Katsuhito Mori,
Takuhito Shoji,
Yohei Mima,
Koka Motoyama,
Shinya Fukumoto,
Atsushi Shioi,
Masanori Emoto, Yasuhiko Yamamoto,
Hiroshi Yamamoto,
Yoshiki Nishizawa,
Masafumi Kurajoh,
Tetsuya Yamamoto,
Masaaki Inaba
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ABSTRACT: Receptor for advanced glycation end products (RAGE) has been shown to be involved in adiposity as well as atherosclerosis even in nondiabetic conditions. In this study, we examined mechanisms underlying how RAGE regulates adiposity and insulin sensitivity. RAGE overexpression in 3T3-L1 preadipocytes using adenoviral gene transfer accelerated adipocyte hypertrophy, whereas inhibitions of RAGE by small interfering RNA significantly decrease adipocyte hypertrophy. Furthermore, double knockdown of high mobility group box-1 and S100b, both of which are RAGE ligands endogenously expressed in 3T3-L1 cells, also canceled RAGE-medicated adipocyte hypertrophy, implicating a fundamental role of ligands-RAGE ligation. Adipocyte hypertrophy induced by RAGE overexpression is associated with suppression of glucose transporter type 4 and adiponectin mRNA expression, attenuated insulin-stimulated glucose uptake, and insulin-stimulated signaling. Toll-like receptor (Tlr)2 mRNA, but not Tlr4 mRNA, is rapidly upregulated by RAGE overexpression, and inhibition of Tlr2 almost completely abrogates RAGE-mediated adipocyte hypertrophy. Finally, RAGE(-/-) mice exhibited significantly less body weight, epididymal fat weight, epididymal adipocyte size, higher serum adiponectin levels, and higher insulin sensitivity than wild-type mice. RAGE deficiency is associated with early suppression of Tlr2 mRNA expression in adipose tissues. Thus, RAGE appears to be involved in mouse adipocyte hypertrophy and insulin sensitivity, whereas Tlr2 regulation may partly play a role.
Diabetes 09/2012; · 8.29 Impact Factor
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ABSTRACT: The structure of a complex between heme(Fe(3+)) and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized by (1)H NMR. The study demonstrated that the heme(Fe(3+)) is sandwiched between the 3'-terminal G-quartets of the G-quadruplex DNA. Hence, the net +1 charge of the heme(Fe(3+)) in the complex is surrounded by the eight carbonyl oxygen atoms of the G-quartets. Interaction between the heme Fe(3+) and G-quartets in the complex was clearly manifested in the solvent (1)H/(2)H isotope effect on the NMR parameters of paramagnetically shifted heme methyl proton signals, and interaction of the heme Fe(3+) with the eight carbonyl oxygen atoms of the two G-quartets was shown to provide a strong and axially symmetric ligand field surrounding the heme Fe(3+), yielding a heme(Fe(3+)) low-spin species with a highly symmetric heme electronic structure. This finding provides new insights as to the design of the molecular architecture and functional properties of various heme-DNA complexes.
Inorganic Chemistry 07/2012; 51(15):8168-76. · 4.60 Impact Factor
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04/2012; , ISBN: 978-953-51-0543-5
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ABSTRACT: Diabetic angiopathy including micro- and macroangiopathy is concerned with high rate of morbidity and mortality in patients with long-standing diabetes. Receptor for advanced glycation end products (RAGE) and its ligands have been considered as important pathogenic triggers for the progression of the vascular injuries in diabetes. The deleterious link between RAGE and diabetic angiopathy has been demonstrated in animal studies. Preventive and therapeutic strategies focusing on RAGE and its ligand axis may be of great importance in relieving diabetic vascular complications and reducing the burden of disease.
Experimental Diabetes Research 01/2012; 2012:894605. · 1.20 Impact Factor
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ABSTRACT: The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3'-terminal G-quartet of the DNA though a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π-π stacking interaction between the pseudo-C₂-symmetric heme and the C₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe-CO bond in the complex is similar to that of the Fe-CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.
European Journal of Biochemistry 12/2011; 17(3):437-45. · 3.42 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa cytochrome c(551) (PA) possesses a long polypeptide loop near its heme, and a unique hydrogen bond network among Ser52, axial Met61, and the heme 13-propionate side chain, i.e., Ser52 amide NH is hydrogen bonded to axial Met61 carbonyl CO, Met61 amide NH to Ser52 carbonyl CO, and Ser52 side chain OH to the heme 13-propionate side chain, contributes to stabilization of the structure of the loop [Y. Matsuura, T. Takano, R.E. Dickerson, J. Mol. Biol. 156 (1982) 389-409]. In this study, the structure and redox function of S52N and S52G mutants were characterized in order to elucidate the role of Ser52 in functional regulation of the protein. We found that the redox function of PA was hardly affected by an S52N mutation, but was slightly by an S52G one. The functional similarity between the wild-type protein and the S52N mutant demonstrated that Asn52 in the mutant plays a similar pivotal role in the formation of the unique hydrogen bond network that stabilizes the structure of the loop as Ser52 in the wild-type protein does. On the other hand, the functional alteration induced by the S52G mutation can be attributed to a structural change of the loop due to the lack of the hydrogen bond between the Gly52 and heme 13-propionate side chain in the mutant. Thus, this study demonstrated that the function of the protein can be tuned through the structural properties of the polypeptide loop near its heme.
Journal of inorganic biochemistry 12/2011; 108:182-7. · 3.25 Impact Factor
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Tomoya Kamide,
Yasuko Kitao,
Toshiaki Takeichi,
Akiko Okada,
Hiromi Mohri,
Ann Marie Schmidt,
Takayuki Kawano,
Seiichi Munesue, Yasuhiko Yamamoto,
Hiroshi Yamamoto,
Jun-ichiro Hamada,
Osamu Hori
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ABSTRACT: The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in a diverse range of pathological conditions. To analyze the roles of RAGE and its decoy receptor, endogenous secretory RAGE (esRAGE), in the global cerebral ischemia, three different mouse cohorts, wild-type, RAGE⁻/⁻, and esRAGE transgenic (Tg) mice were subjected to bilateral common carotid artery occlusion (BCCAO). RT-PCR and immunohistochemical analysis revealed that expression of RAGE was induced in the vascular cells at 12 h, and then in the neurons and glia from 3 to 7 days in the hippocampus after BCCAO. The numbers of surviving neurons in the hippocampal CA1 region were significantly higher in RAGE⁻/⁻ and esRAGE Tg mice than those in wild-type mice in the periods between 24 h and 7 days after BCCAO. Lower levels of 3-nitrotyrosine (3-NT) and higher levels of endothelial nitric oxide synthase (eNOS), together with enlarged vascular areas were observed in RAGE⁻/⁻ and esRAGE Tg mice at 12 h after BCCAO. In the later periods, expressions of glia-derived inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS) were reduced in RAGE⁻/⁻ and esRAGE Tg mice. These results suggest that RAGE may contribute to delayed neuronal death after global cerebral ischemia by enhancing vascular injury and deleterious glia-mediated inflammation.
Neurochemistry International 12/2011; 60(3):220-8. · 2.86 Impact Factor
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ABSTRACT: Circulating endothelial progenitor cells (EPCs) derived from the bone marrow (BM) participate in maintaining endothelial integrity and vascular homeostasis. Reduced EPC number and function result in vascular complications in diabetes. EPCs are a population of cells existing in various differentiation stages, and their cell surface marker profiles change during the process of mobilization and maturation. Hence, a generally accepted marker combination and a standardized protocol for the quantification of EPCs remain to be established. To determine the EPC subsets that are affected by diabetes, we comprehensively analyzed 32 surface marker combinations of mouse peripheral blood (PB), BM, and spleen cells by multicolor flow cytometry. Ten subsets equivalent to previously reported mouse EPCs significantly declined in number in the PB of streptozotocin-induced diabetic mice, and this reduction was reversed by insulin treatment. The PI(-)Lin(-)c-Kit(-)Sca-1(+)Flk-1(-)CD34(-)CD31(+) EPC cluster, which can differentiate into mature endothelial cells in vitro, was the highest population in the PB, BM, and spleen and occurred 61 times more in the spleen than in the PB. The cell number significantly decreased in the BM as well as in the PB but paradoxically increased in the spleen under diabetic conditions. Insulin treatment reversed the decrease of EPC subsets in the BM and PB and reversed their increase in spleen. A similar tendency was observed in some of the major cell populations in db/db mice. To the best of our knowledge, we are the first to report spatial population changes in mouse EPCs by diabetes in the blood and in the BM across the spleen. Diminished circulating EPC supply by diabetes may be ascribed to impaired EPC production in the BM and to decreased EPC mobilization from the spleen, which may contribute to vascular dysfunction in diabetic conditions.
AJP Cell Physiology 12/2011; 302(6):C892-901. · 3.54 Impact Factor
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Takahiro Sugihara,
Seiichi Munesue, Yasuhiko Yamamoto,
Shigeru Sakurai,
Nasima Akhter,
Yoji Kitamura,
Kazuhiro Shiba,
Takuo Watanabe,
Hideto Yonekura,
Yasuhiko Hayashi,
Jun-Ichiro Hamada,
Hiroshi Yamamoto
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ABSTRACT: The cell-surface receptor for advanced glycation end-products (RAGE) has been implicated in the development of diabetic vascular complications and Alzheimer's disease. RAGE has been considered to be involved in amyloid-β1-42 (Aβ1-42) uptake into brain. In the present study, we demonstrate that endogenous secretory RAGE (esRAGE), a decoy form of RAGE generated by alternative RNA processing, is able to inhibit Aβ1-42 influx into mouse brain. Surface plasmon resonance and competitive binding assays revealed that human Aβ1-42 interacted with human esRAGE within the immunoglobulin V type region. We next examined the uptake and distribution of 125I-labeled human Aβ1-42 in various organs and body fluids of newly created mice overexpressing human esRAGE as well as RAGE-null and wild-type (WT) mice. The transition of the 125I-labeled Aβ1-42 from circulation to brain parenchyma peaked at 30 min after the injection into WT mice, but this was significantly blunted in esRAGE-overexpressing and RAGE-null mice. Significant reduction in 125I-labeled Aβ1-42-derived photo-stimulated luminescence were marked in ventricles, cerebral cortex, hippocampus, especially CA1 and CA3 regions, putamen, and thalamus. The results thus suggest the potential of esRAGE in protection against the development of Alzheimer's disease.
Journal of Alzheimer's disease: JAD 11/2011; 28(3):709-20. · 3.74 Impact Factor
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ABSTRACT: Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c(552) (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T(m)) value by ∼ 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the Val/Ile mutant exhibited a negative shift of ∼ 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by ∼ 3 and ∼ 5 deg, respectively, and the E(m) value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E(m) value of the Val/Leu mutant could be reasonably interpreted in terms of the different effects of the mutation on the stabilities of the two different redox forms of the protein. Thus, the present study demonstrated that the stability of the protein is affected quite sensitively by the contextual stereochemical packing of hydrophobic residues in the protein interior and that the structural properties of the hydrophobic core in the protein interior are crucial for control of the redox function of the protein. These findings provide novel insights as to functional control of a protein, which could be utilized for tuning of the T(m) and E(m) values of the protein by means of protein engineering.
Biochemistry 03/2011; 50(15):3161-9. · 3.42 Impact Factor
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Yasuhiko Yamamoto,
Ai Harashima,
Hidehito Saito,
Koichi Tsuneyama,
Seiichi Munesue,
So Motoyoshi,
Dong Han,
Takuo Watanabe,
Masahide Asano,
Shin Takasawa,
Hiroshi Okamoto,
Satoshi Shimura,
Tadahiro Karasawa,
Hideto Yonekura,
Hiroshi Yamamoto
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ABSTRACT: Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.
The Journal of Immunology 03/2011; 186(5):3248-57. · 5.79 Impact Factor
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Nippon rinsho. Japanese journal of clinical medicine 11/2010; 68 Suppl 9:39-44.
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ABSTRACT: In the denatured state of Hydrogenobacter thermophilus cytochrome c(552) (HT), the N-terminal amino group of the polypeptide chain is coordinated to the heme Fe in place of the axial Met, the His-N(term) form being formed [Tai, H., Munegumi, T., Yamamoto, Y. Inorg. Chem. 2009, 48, 331-338]. Since the His-N(term) form can be considered as an ordered residual structure in the denatured protein, its stability significantly influences the energy of the denatured state. In this study, the His-N(term) forms of the wild-type HT and its mutants possessing a series of amino acid residues at the N-terminal, such as N1D, N1E, and N1G, have been characterized to elucidate the physicochemical properties of the N-terminal residue responsible for the control of the thermodynamic stability of the His-N(term) form. The study revealed that the thermodynamic stability of the His-N(term) form depends highly on the basicity of the N-terminal amino group of the polypeptide chain in such a manner that an increase in the pK(a) value of the N-terminal amino group by 1 unit results in stabilization of the bond between heme Fe and the N-terminal amino group (Fe-N(term) bond) in the His-N(term) form by ∼4 kJ mol(-1). The empirical hard and soft acid and base principle could account for the observed relationship between the pK(a) value of the N-terminal amino group and the stability of the Fe-N(term) bond in the His-N(term) form. In addition, the study demonstrated that the overall stability of the protein can be manipulated through the energy of the denatured protein by changing the thermodynamic stability of the His-N(term) form. Consequently, the overall stability of the protein has been shown to be controlled through alteration of the basicity of the N-terminal amino group of the polypeptide chain. These findings provide new insights into the stabilizing interactions in the denatured protein, which are relevant as to characterization of the protein stability and folding.
Inorganic Chemistry 11/2010; 49(23):10840-6. · 4.60 Impact Factor
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ABSTRACT: Human adult haemoglobin (Hb A), a tetrameric oxygen transfer haemoprotein, has been recognized as an excellent model for investigating the structure-function relationships in allosteric proteins, and has been characterized exhaustively from both experimental and theoretical aspects. Despite the detailed structural and spectroscopic information available for the protein, functional properties have not been as fully elucidated as expected, and hence have remained unexplored. A major drawback for the functional characterization of Hb A is the lack of experimental techniques which enable quantitative characterization of functional properties of the individual subunits of the intact protein. In this study, we have developed techniques for determining the equilibrium constant of the acid-alkaline transition, usually represented as the 'pK(a)' value, in the individual subunits of the met-forms of Hb A (metHb A) and human foetal haemoglobin (metHb F). The pK(a) values of the individual subunits of metHb A and metHb F have been shown to constitute novel and highly sensitive probes for characterizing the effects of structural changes of not only the interfaces between the subunits within the protein, but also the contact between haem and the protein in the haem pocket. In addition, haem replacement studies of the proteins revealed that the contact between the haem peripheral vinyl side chain and the protein in the haem pocket is important for maintaining the non-equivalence in the haem environment between the subunits of Hb A and Hb F, which could be relevant to the cooperative ligand binding of the proteins.
Journal of biochemistry 08/2010; 148(2):217-29. · 1.95 Impact Factor
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Tomokazu Shibata,
Satoshi Nagao,
Masashi Fukaya,
Hulin Tai,
Shigenori Nagatomo,
Kenji Morihashi,
Takashi Matsuo,
Shun Hirota,
Akihiro Suzuki,
Kiyohiro Imai, Yasuhiko Yamamoto
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ABSTRACT: Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.
Journal of the American Chemical Society 04/2010; 132(17):6091-8. · 9.91 Impact Factor
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ABSTRACT: Receptor for advanced glycation endproducts (RAGE) is a cell-surface receptor. The binding of ligands to membrane-bound RAGE (mRAGE) evokes cellular responses involved in various pathological processes. Previously, we identified a novel soluble form, endogenous secretory RAGE (esRAGE) generated by alternative 5' splice site selection in intron 9 that leads to extension of exon 9 (exon 9B). Because esRAGE works as an antagonistic decoy receptor, the elucidation of regulatory mechanism of the alternative splicing is important to understand RAGE-related pathological processes. Here, we identified G-rich cis-elements within exon 9B for regulation of the alternative splicing using a RAGE minigene. Mutagenesis of the G-rich cis-elements caused a drastic increase in the esRAGE/mRAGE ratio in the minigene-transfected cells and in loss of binding of the RNA motif to heterogenous nuclear ribonucleoprotein (hnRNP) H. On the other hand, the artificial introduction of a G-stretch in exon 9B caused a drastic decrease in the esRAGE/mRAGE ratio accompanied by the binding of hnRNP H to the RNA motif. Thus, the G-stretches within exon 9B regulate RAGE alternative splicing via interaction with hnRNP H. The findings should provide a molecular basis for the development of medicines for RAGE-related disorders that could modulate esRAGE/mRAGE ratio.
Journal of biochemistry 12/2009; 147(5):651-9. · 1.95 Impact Factor
