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ABSTRACT: Polyglutamine tract-binding protein 1 (PQBP1) is a nuclear protein that regulates transcription and pre-mRNA splicing. In addition, the mutations in the PQBP1 gene are known to cause hereditary mental retardation. This review summarizes current knowledge about the solution structure of PQBP1. PQBP1 is an intrinsically disordered protein: its polar-rich domain and C-terminal domain are disordered under physiological conditions. PQBP1 binds to its target molecule U5-15kD via a continuous 23-residue segment of the C-terminal domain. The function of PQBP1 in the pre-mRNA splicing is also discussed.
YAKUGAKU ZASSHI 01/2013; 133(5):519-26. · 0.37 Impact Factor
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ABSTRACT: The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.
Biochemistry 07/2012; 51(31):6089-96. · 3.42 Impact Factor
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Hirofumi Jono,
Takayuki Anno,
Keiichi Motoyama,
Yohei Misumi,
Masayoshi Tasaki,
Toshinori Oshima,
Yoshimasa Mori, Mineyuki Mizuguchi,
Mitsuharu Ueda,
Satoru Shinriki,
Makoto Shono,
Konen Obayashi,
Hidetoshi Arima,
Yukio Ando
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ABSTRACT: Transthyretin (TTR)-related familial amyloidotic polyneuropathy, which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils. Although it is believed that protein misfolding of monomeric form of TTR is a rate-limiting step for TTR amyloid formation, no effective therapy targeting this misfolding step is available. Our recent studies revealed that cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, might interact with TTR and prevent the protein misfolding. In this study, we focused on and elucidated the inhibitory effect of 6-O-α-(4-O-α-D-Glucuronyl)-D-glucosyl-β-CyD (GUG-β-CyD) on TTR amyloid formation. Tryptophan (Trp) fluorescence and (1)H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized TTR conformation via interaction with the hydrophobic amino acids of TTR. Moreover, GUG-β-CyD suppressed TTR deposition in transgenic rats possessing a human ATTR V30M gene in vivo. Collectively, these data indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.
Amyloid: the international journal of experimental and clinical investigation: the official journal of the International Society of Amyloidosis 06/2012; 19 Suppl 1:50-2. · 2.12 Impact Factor
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Mitsuharu Ueda,
Naohide Ageyama,
Shinichiro Nakamura,
Minami Nakamura,
James Kenn Chambers,
Yohei Misumi, Mineyuki Mizuguchi,
Satoru Shinriki,
Satomi Kawahara,
Masayoshi Tasaki,
Hirofumi Jono,
Konen Obayashi,
Erika Sasaki,
Yumi Une,
Yukio Ando
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ABSTRACT: Mutant forms of transthyretin (TTR) cause the most common type of autosomal-dominant hereditary systemic amyloidosis. In addition, wild-type TTR causes senile systemic amyloidosis, a sporadic disease seen in the elderly. Although spontaneous development of TTR amyloidosis had not been reported in animals other than humans, we recently determined that two aged vervet monkeys (Chlorocebus pygerythrus) spontaneously developed systemic TTR amyloidosis. In this study here, we first determined that aged vervet monkeys developed TTR amyloidosis and showed cardiac dysfunction but other primates did not. We also found that vervet monkeys had the TTR Ile122 allele, which is well known as a frequent mutation-causing human TTR amyloidosis. Furthermore, we generated recombinant monkey TTRs and determined that the vervet monkey TTR had lower tetrameric stability and formed more amyloid fibrils than did cynomolgus monkey TTR, which had the Val122 allele. We thus propose that the Ile122 allele has an important role in TTR amyloidosis in the aged vervet monkey and that this monkey can serve as a valid pathological model of the human disease. Finally, from the viewpoint of molecular evolution of TTR in primates, we determined that human TTR mutations causing the leptomeningeal phenotype of TTR amyloidosis tended to occur in amino acid residues that showed no diversity throughout primate evolution. Those findings may be valuable for understanding the genotype-phenotype correlation in this inherited human disease.
Laboratory Investigation 12/2011; 92(3):474-84. · 3.64 Impact Factor
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Hirofumi Jono,
Takayuki Anno,
Keiichi Motoyama,
Yohei Misumi,
Masayoshi Tasaki,
Toshinori Oshima,
Yoshimasa Mori, Mineyuki Mizuguchi,
Mitsuharu Ueda,
Makoto Shono,
Konen Obayashi,
Hidetoshi Arima,
Yukio Ando
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ABSTRACT: TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.
Biochemical Journal 07/2011; 437(1):35-42. · 4.90 Impact Factor
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Takahide Kouno,
Nobuhisa Watanabe,
Naoki Sakai,
Takashi Nakamura,
Yuko Nabeshima,
Masashi Morita, Mineyuki Mizuguchi,
Tomoyasu Aizawa,
Makoto Demura,
Tsuneo Imanaka,
Isao Tanaka,
Keiichi Kawano
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ABSTRACT: Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.
Journal of Molecular Biology 01/2011; 405(2):560-9. · 4.00 Impact Factor
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Yasuhiro Nonaka,
Hideki Muto,
Tomoyasu Aizawa,
Etsuro Okabe,
Shohei Myoba,
Takuya Yokoyama,
Shin Saito,
Fumie Tatami,
Yasuhiro Kumaki,
Masakatsu Kamiya,
Takashi Kikukawa, Mineyuki Mizuguchi,
Shigeharu Takiya,
Masataka Kinjo,
Makoto Demura,
Keiichi Kawano
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ABSTRACT: The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.
Biochemistry 09/2010; 49(38):8367-75. · 3.42 Impact Factor
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Masanori Miyata,
Takashi Sato,
Miyuki Kugimiya,
Misato Sho,
Teruya Nakamura,
Shinji Ikemizu,
Mami Chirifu, Mineyuki Mizuguchi,
Yuko Nabeshima,
Yoshiaki Suwa,
Hiroshi Morioka,
Takao Arimori,
Mary Ann Suico,
Tsuyoshi Shuto,
Yasuhiro Sako,
Mamiko Momohara,
Tomoaki Koga,
Saori Morino-Koga,
Yuriko Yamagata,
Hirofumi Kai
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ABSTRACT: Amyloid fibril formation is associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Familial amyloid polyneuropathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T(4)). TTR variants contribute to the pathogenesis of amyloidosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigallocatechin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TTR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V30M TTR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy.
Biochemistry 07/2010; 49(29):6104-14. · 3.42 Impact Factor
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Masakatsu Kamiya,
Keisuke Oyauchi,
Yoshinori Sato,
Takuya Yokoyama,
Mofei Wang,
Tomoyasu Aizawa,
Yasuhiro Kumaki, Mineyuki Mizuguchi,
Kunio Imai,
Makoto Demura,
Koichi Suzuki,
Keiichi Kawano
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ABSTRACT: We previously reported that yamamarin, a pentapeptide with an amidated C-terminus (DILRG-NH(2)) isolated from larvae of the silkmoth, and its palmitoylated analog (C16-DILRG-NH(2)) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure-activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C-terminal part (-RG-NH(2)) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG-NH(2) and C16-DILRG-NH(2) revealed that the N-terminal palmitoyl group of C16-DILRG-NH(2) did not affect the conformation of the C-terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs.
Journal of Peptide Science 05/2010; 16(5):242-8. · 1.80 Impact Factor
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ABSTRACT: Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts with various proteins, including RNA polymerase II and the spliceosomal protein U5-15kD. PQBP-1 is known to be associated with X-linked mental retardation in which a frameshift mutation in the PQBP-1 gene occurs. In the present study, we demonstrate that PQBP-1 binds to U5-15kD via a continuous 23-residue segment within its C-terminal domain. Intriguingly, this segment is lost in the frameshift mutants of PQBP-1 associated with X-linked mental retardation. These findings suggest that the frameshift mutations in the PQBP-1 gene lead to expression of mutants lacking the ability to interact with U5-15kD.
Biochimica et Biophysica Acta 03/2010; 1804(7):1500-7. · 4.66 Impact Factor
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Masanori Miyata,
Takashi Sato, Mineyuki Mizuguchi,
Teruya Nakamura,
Shinji Ikemizu,
Yuko Nabeshima,
Seiko Susuki,
Yoshiaki Suwa,
Hiroshi Morioka,
Yukio Ando,
Mary Ann Suico,
Tsuyoshi Shuto,
Tomoaki Koga,
Yuriko Yamagata,
Hirofumi Kai
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ABSTRACT: Transthyretin (TTR) is a tetrameric protein associated with amyloidosis caused by tetramer dissociation and monomer misfolding. The structure of two TTR variants (E54G and E54K) with Glu54 point mutation that cause clinically aggressive amyloidosis remains unclear, although amyloidogenicity of artificial triple mutations (residues 53-55) in beta-strand D had been investigated. Here we first analyzed the crystal structures and biochemical and biophysical properties of E54G and E54K TTRs. The direction of the Lys15 side chain in E54K TTR and the surface electrostatic potential in the edge region in both variants were different from those of wild-type TTR. The presence of Lys54 leads to destabilization of tetramer structure due to enhanced electrostatic repulsion between Lys15 of two monomers. Consistent with structural data, the biochemical analyses demonstrated that E54G and E54K TTRs were more unstable than wild-type TTR. Furthermore, the entrance of the thyroxine (T(4)) binding pocket in TTR was markedly narrower in E54K TTR and wider in E54G TTR compared with wild-type TTR. The tetramer stabilization and amyloid fibril formation assays in the presence of T(4) showed lower tetramer stability and more fibril formation in E54K and E54G TTRs than in wild-type TTR, suggesting decreased T(4) binding to the TTR variants. These findings indicate that structural modification by Glu54 point mutation may sufficiently alter tetramer stability and T(4) binding.
Biochemistry 12/2009; 49(1):114-23. · 3.42 Impact Factor
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ABSTRACT: Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino
acid GBP (1–23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid
GBP (1–28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants,
which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional
structures. The results of an injection study indicate that 1–28 GBP most strongly suppresses larval growth. NMR analysis
shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered.
However, the C-terminal region of 1–28 GBP undergoes a conformational transition from a random coiled state to an α-helical
state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect
larval growth activity.
Journal of Biological Chemistry 10/2009; 284(43):29625-29634. · 4.77 Impact Factor
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ABSTRACT: Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.
Journal of Biological Chemistry 09/2009; 284(43):29625-34. · 4.77 Impact Factor
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ABSTRACT: Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. The amino acid sequence of big defensin is divided into an N-terminal hydrophobic domain and a C-terminal cationic domain, which are responsible for antimicrobial activities against Gram-positive and -negative bacteria, respectively. The N-terminal domain of big defensin forms a unique globular conformation with two alpha-helices and a parallel beta-sheet, while the C-terminal domain adopts a beta-defensin-like fold. Although our previous study implied that big defensin changes its N-terminal structure in a micellar environment, due to the poor quality of the NMR spectra it remained to be resolved whether the N-terminal domain adopts any structure in the presence of micelles. In this analysis, we successfully determined the structure of the N-terminal fragment of big defensin in a micellar solution, showing that the fragment peptide forms a single alpha-helix structure. Moreover, NMR experiments using paramagnetic probes revealed that the N-terminal domain of big defensin penetrates into the micelle with a dipping at the N-terminal edge of the alpha-helix. Here, we propose a model for how big defensin associates with the target membrane.
Biochemistry 08/2009; 48(32):7629-35. · 3.42 Impact Factor
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Mostafa M Sira,
Taketoshi Yoshida,
Makoto Takeuchi,
Yoshinori Kashiwayama,
Takeshi Futatani,
Hirokazu Kanegane,
Akiko Sasahara,
Yasunori Ito, Mineyuki Mizuguchi,
Tsuneo Imanaka,
Toshio Miyawaki
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ABSTRACT: Human colostrum contains many bioactive factors that must promote the development of intestinal mucosal immunity in infants. Especially, the presence of certain cytokines such as transforming growth factor (TGF)-beta or IL-10 has been of great interest for IgA production as a function of mucosal immune response. In the present study, we attempted to investigate whether unidentified factors inducing generation of IgA-producing cells from naive B cells might exist in colostrum. For this purpose, colostrum samples were directly added to a culture consisting of naive B cells and dendritic cells from cord blood and CD40 ligand-transfected L cells, comparing with recombinant IL-10 (rIL-10) and/or rTGF-beta. It was noted that most colostrum samples alone were able to induce IgA-secreting cells at higher levels than rIL-10 and/or rTGF-beta. IgA-inducing activity of colostrum was abolished by neither anti-neutralizing mAbs against IL-10 nor TGF-beta, though partially by anti-IL-6 mAb. We prepared partially purified fractions from both pooled colostrums with and without IgA-inducing activity and comparatively performed quantitative proteomic analysis by two-dimensional difference gel electrophoresis followed by liquid chromatography-mass spectrometry. As a result, syntenin-1 was identified as a candidate for IgA-inducing protein in colostrum. Western blot analysis indicated that levels of syntenin-1 in colostrum samples were correlated with their IgA-inducing activities. Moreover, we demonstrated that recombinant syntenin-1 could induce preferentially IgA production from naive B cells. These results suggest that syntenin-1 serves as one of IgA-inducing factors for B cells.
International Immunology 08/2009; 21(9):1013-23. · 3.41 Impact Factor
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ABSTRACT: In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.
FEBS Journal 05/2009; 276(8):2192-200. · 3.79 Impact Factor
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Mitsuharu Ueda,
Yohei Misumi, Mineyuki Mizuguchi,
Masaaki Nakamura,
Taro Yamashita,
Yoshiki Sekijima,
Kazutoshi Ota,
Satoru Shinriki,
Hirofumi Jono,
Shu-ichi Ikeda,
Ole B Suhr,
Yukio Ando
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ABSTRACT: Mass spectrometric analyses are valuable for detection of transthyretin (TTR) variants, which cause familial amyloidotic polyneuropathy (FAP). However, those methods require an immunoprecipitation step with an anti-TTR antibody and are not suitable for quantitative detection. We investigated the usefulness of SELDI-TOF mass spectrometry (MS) without an immunoprecipitation step.
We used ProteinChips with chromatographic capture formats to detect TTRs. We attempted to correlate the intensity of mixed samples of amyloidogenic TTR (ATTR) V30M to wild-type (WT) TTR. We analyzed the proportion of ATTR V30M in amyloid-laden cardiac tissues from FAP patients, and also evaluated samples from FAP patients with 16 other TTR mutations.
Detection of ATTR required only 3 h of SELDI-TOF MS analysis. We determined that SELDI-TOF MS was suitable for quantitative detection of ATTR V30M and demonstrated that the proportion of ATTR V30M to WT TTR was 46.6% in amyloid-laden cardiac tissue from an FAP patient who died 10 years after liver transplantation. With this method, we identified 12 of 17 TTR variants. Small mass shifts and low concentrations of variants prevented ATTR detection. By changing the analytical conditions, we achieved detection of low concentrations of ATTR Y114C in serum.
SELDI-TOF MS is a reliable tool for quantitative evaluation of TTR variants, in both tissue amyloid deposits and body fluids. This method is useful for the diagnosis and investigation of the pathogenesis of FAP.
Clinical Chemistry 05/2009; 55(6):1223-7. · 7.91 Impact Factor
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ABSTRACT: Polyglutamine tract binding protein-1 (PQBP-1) is a nuclear protein that interacts with disease proteins containing expanded polyglutamine repeats. PQBP-1 also interacts with RNA polymerase II and a spliceosomal protein U5-15kD. In the present study, we demonstrate that PQBP-1 is composed of a large unstructured region and a small folded core. Intriguingly, the large unstructured region encompasses two functional domains: a polar amino acid rich domain and a C-terminal domain. These findings suggest that PQBP-1 belongs to the family of intrinsically unstructured/disordered proteins. Furthermore, the binding of the target molecule U5-15kD induces only minor conformational changes into PQBP-1. Our results suggest that PQBP-1 includes high content of unstructured regions in the C-terminal domain, in spite of the binding of U5-15kD.
Biochimica et Biophysica Acta 04/2009; 1794(6):936-43. · 4.66 Impact Factor
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Tatsuro Kamijima,
Ayaka Ohmura,
Toshiya Sato,
Kaoru Akimoto,
Miki Itabashi, Mineyuki Mizuguchi,
Masakatsu Kamiya,
Takashi Kikukawa,
Tomoyasu Aizawa,
Masayuki Takahashi,
Keiichi Kawano,
Makoto Demura
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ABSTRACT: HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.
Biochemical and Biophysical Research Communications 10/2008; 376(1):211-4. · 2.48 Impact Factor
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ABSTRACT: Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a beta-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.
Biochemistry 10/2008; 47(40):10611-9. · 3.42 Impact Factor
