Publications (13)38.53 Total impact
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Article: Species-barrier phenomenon in prion transmissibility from a viewpoint of protein science.
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ABSTRACT: Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal infectious neurodegenerative disorders. Their causative agents are prions which are composed of disease-associated forms of prion protein (PrP(Sc)). Naturally occurring cases of TSEs are found in several mammalian species including humans, sheep, goats, minks, cattle and deer. Prions are also experimentally transmissible to other mammals such as mice, hamsters and monkeys, but interspecies transmission is often inefficient due to the 'species-barrier'. Studies have suggested that the barrier is not simply determined by differences in amino acid sequences of cellular prion protein (PrP(C)) among animal species, but also by prion strains which are closely associated with conformational properties of PrP(Sc) aggregates. Although the conformational properties of PrP(Sc) remain largely unknown, recent investigation of local structures of PrP(C) and, in particular, structural modeling of PrP(Sc) aggregates have provided molecular insight into this field. In this review, we discuss the species-barrier phenomenon in terms of the protein science.Journal of biochemistry 01/2013; · 1.95 Impact Factor -
Article: Spatial arrangement of rhodopsin in retinal rod outer segment membranes studied by spin-labeling and pulsed electron double resonance.
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ABSTRACT: We have determined the spatial arrangement of rhodopsin in the retinal rod outer segment (ROS) membrane by measuring the distances between rhodopsin molecules in which native cysteines were spin-labeled at ~1.0 mol/mol rhodopsin. The echo modulation decay of pulsed electron double resonance (PELDOR) from spin-labeled ROS curved slightly with strong background decay. This indicated that the rhodopsin was densely packed in the retina and that the rhodopsin molecules were not aligned well. The curve was simulated by a model in which rhodopsin is distributed randomly as monomers in a planar membrane.Biochemical and Biophysical Research Communications 07/2012; 425(2):134-7. · 2.48 Impact Factor -
Article: Mouse prion protein (PrP) segment 100 to 104 regulates conversion of PrP(C) to PrP(Sc) in prion-infected neuroblastoma cells.
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ABSTRACT: Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).Journal of Virology 03/2012; 86(10):5626-36. · 5.40 Impact Factor -
Article: Correction to "Photoinduced Rearrangement of Aromatic N-Chloroamides to Chloroaromatic Amides in the Solid State. Inverted Π(N)-Σ(N) Occupational Stability of Amidyl Radicals"
The Journal of Physical Chemistry A 06/2011; · 2.95 Impact Factor -
Article: Photoinduced rearrangement of aromatic N-chloroamides to chloroaromatic amides in the solid state: inverted Π(N)-Σ(N) occupational stability of amidyl radicals.
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ABSTRACT: We report a solid-state photochemical rearrangement reaction by which aromatic N-chloroamides exposed to UV light or sunlight are rapidly and efficiently converted to chloroaromatic amides. The course, the intermediate (nascent chlorine vs dichlorine) and the outcome of the reaction depend on the excitation (exposure time, wavelength, and intensity) and on inherent structural factors (the directing role of the substituents and, as demonstrated by the different reactivity of two polymorphs of N-chlorobenzanilide, the supramolecular structure). The photolysis of the chloroamides provides facile photochemical access to arylamidyl radicals as intermediates, which in the absence of strong hydrogen bond donors are stabilized in the reactant crystals by C-H/N-Cl···π interactions, thus, providing insight into their structure and chemistry. Thorough theoretical modeling of the factors determinant to the stability and the nature of the spin-hosting orbital evidenced that although the trans-Π(||) state (Np spin) of the amidyls is normally preferred over the trans-Σ(⊥) configuration (Nsp(2) spin), stabilization by aromatic conjugation, steric and geometry factors, as well as by electronic effects from the substituents can decrease the Π-Σ gap in these intermediates significantly, resulting in similar and, in the case of the orthogonal amide-phenyl disposition, even reversed population of the unpaired electron in the two orbitals. Quantitative correlation established that the inverted occupational spin stability and the Π(N)-Σ(N) crossover are collectively facilitated by the conformation, valence angle, and disposition of the amide group relative to the aromatic system. The stabilization and detection of a trans-Σ(⊥) radical was experimentally accomplished by steric locking of the orthogonal trans-amide conformation with double ortho-tert-butyl substitution at the phenyl ring. The effects of the single para-phenyl substituents on the relative occupational stability of the arylamidyl radical states point out to non-Hammett behavior. By including cumulative electronic effects from multiple substitutions, four distinct families of the aromatic amidyl radicals were identified. The Π(∥) state is the most stable structure of the N-phenylacetamidyl radical and of most of the substituted arylamidyls, although the Σ(⊥) and Π(⊥) states can also be stabilized by introducing tert-butyl and nitro groups, respectively.The Journal of Physical Chemistry A 06/2011; 115(26):7834-48. · 2.95 Impact Factor -
Article: Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases.
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ABSTRACT: Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood. In a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-ΔC in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-ΔC in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease. We identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders.Proteome Science 10/2010; 8:53. · 2.33 Impact Factor -
Article: An improved method for cell-to-cell transmission of infectious prion.
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ABSTRACT: Prion diseases are characterized by the accumulation of a pathological form of prion protein (PrP(Sc)), which behaves as an infectious agent. Here we developed an in vitro co-culture system to analyze the PrP(Sc) transmission from ScN2a cell, which persistently retains PrP(Sc), to naïve N2a cell. In this cell-to-cell transmission system, PrP(Sc) transmitted to recipient N2a cell was able to be detected within 5-7days. Further characterization showed that higher cell density greatly facilitated the transmission of PrP(Sc). This improved in vitro transmission method may become a useful tool for unveiling the molecular mechanism of PrP(Sc) transmission.Biochemical and Biophysical Research Communications 07/2010; 397(3):505-8. · 2.48 Impact Factor -
Article: Switch action of troponin on muscle thin filament as revealed by spin labeling and pulsed EPR.
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ABSTRACT: We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys(133) of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca(2+) state, the TnC44-TnI133-T distance was 42 A, with a narrow distribution (half-width of 9 A), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 A (width of 12 A) and 22 A (width of 3.4 A), respectively. These values were all consistent with recently published crystal structure (Vinogradova, M. V., Stone, D. B., Malanina, G. G., Karatzaferi, C., Cooke, R., Mendelson, R. A., and Fletterick, R. J. (2005) Proc. Natl Acad. Sci. U.S.A. 102, 5038-5043). Similar distances were obtained with the same spin pairs on a reconstituted thin filament in the +Ca(2+) state. In the -Ca(2+) state, the distances displayed broad distributions, suggesting that the regulatory region of TnI was physically released from the N-lobe of TnC and consequently fluctuated over a variety of distances on a large scale (20-80 A). The interspin distance appeared longer on the filament than on troponin alone, consistent with the ability of the region to bind actin. These results support a concept that the regulatory region of TnI, as a molecular switch, binds to the exposed hydrophobic patch of TnC and traps the inhibitory region of TnI away from actin in Ca(2+) activation of muscle.Journal of Biological Chemistry 02/2010; 285(14):10671-7. · 4.77 Impact Factor -
Article: Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases
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ABSTRACT: Abstract Background Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood. Results In a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-ΔC in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-ΔC in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease. Conclusions We identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders.Proteome Science. 01/2010; -
Article: Direct observation of aminyl radical intermediate during single-crystal to single-crystal photoinduced Orton rearrangement.
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ABSTRACT: A photoinduced analogue of the thermal Orton rearrangement reaction by which an N-chlorine atom from a side amino group is transferred to a phenyl ring was studied in the solid state. Contrary to the mixture of products obtained in solution, in the N-chloro-N-acetylaminobenzene crystals the photoreaction proceeds with complete preservation of crystallinity, affording selectively and quantitatively the para isomer of chloroacetanilide. Study of the reaction mechanism by in situ steady-state photodiffraction, a combination of photoexcitation by UV light and single-crystal X-ray diffraction analysis, provided evidence for creation of N-acetyl-N-phenylaminyl (AcPhN*) radical as a metastable reaction intermediate. The structure of the aminyl radical produced in 9.2% yield from the major disordered component in the statically 85.6:14.4 disordered crystal was directly observed for the first time. The unprecedented stability of the radical is prescribed to the solid-state cage effect, the reactive center of the radical species being locked away from the reactive target molecules. The creation of the radical and its head-to-tail chain reaction within the undulated hydrogen-bonded ribbons involving the acetyl carbonyl group are employed to explain the high selectivity of the photoinduced single-crystal to single-crystal Orton rearrangement. On the basis of the change of the crystal structure and the physicochemical data, a three-center five-atom mechanism involving homolytic cleavage of the N-Cl bond followed by hydrogen abstraction by the carbonyl group is suggested for the solid-state photoinduced Orton rearrangement.The Journal of Physical Chemistry B 10/2007; 111(35):10373-8. · 3.70 Impact Factor -
Article: A systematic evaluation of the function of the protein-remodeling factor Hsp104 in [PSI+] prion propagation in S. cerevisiae by comprehensive chromosomal mutations.
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ABSTRACT: The yeast prion [PSI(+)] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI(+)] propagation, however the structure-and-function relationship of Hsp104 for [PSI(+)] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI(+)] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI(+)] propagation. However, nine mutants were impaired exclusively for [PSI(+)], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI(+)] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI(+)] prion aggregates and that the lateral channel plays a role specific to [PSI(+)] prion propagation.Prion 02/2007; 1(1):69-77. · 2.85 Impact Factor -
Article: Calcium structural transition of human cardiac troponin C in reconstituted muscle fibres as studied by site-directed spin labelling.
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ABSTRACT: The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca(2+)-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32ns to 13ns upon Ca(2+)-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16ns) and immobile (35ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. We determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstroms and 27.2 angstroms in the monomer state and in fibres, respectively, and the addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state.Journal of Molecular Biology 05/2005; 348(1):127-37. · 4.00 Impact Factor -
Article: Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission.
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ABSTRACT: The yeast [PSI+] factor is transmitted by a prion mechanism involving self-propagating Sup35 aggregates. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI+], contains two species-signature elements-a Gln/Asn-rich region (residues 1-41; designated NQ) that is followed by oligopeptide repeats (designated NR). In this study, we show that S. cerevisiae[PSI+] is transmissible through plasmid shuffling and cytoplasmic transfer to heterotypic Sup35s whose NQ is replaced with the S. cerevisiae NQ. In addition to homology, the N-terminal location is essential for NQ mediated susceptibility to [PSI+] transmission amongst heterotypic Sup35s. In vitro, a swap of NQ of S. cerevisiae Sup35 led to cross seeding of amyloid formation. These findings suggest that NQ discriminates self from non-self, and is sufficient to initiate [PSI+] transmission irrespective of whether NR is heterotypic. NR as well as NQ alone coalesces into existing [PSI+] aggregates, showing their independent potentials to interact with the identical sequence in the [PSI+] conformer. The role of NQ and NR in [PSI+] prion formation is discussed.Genes to Cells 01/2004; 8(12):925-39. · 2.68 Impact Factor
Top co-authors
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Institutions
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2012
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National Institute of Infectious Diseases, Tokyo
Tokyo, Tokyo-to, Japan
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2005–2012
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Osaka University
- • Biological Science
- • Department of Beam Materials Science
- • Department of Biological Sciences
Ōsaka-shi, Osaka-fu, Japan
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2004
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The University of Tokyo
- Institute of Medical Science
Tokyo, Tokyo-to, Japan
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