PubMed Health⌕ Search

Biomedical subjects

Yoshinori Fujiyoshi

Publications and source records attributed to Yoshinori Fujiyoshi.

At least 19 recordsLinked to original sources
PubMed2006

Aquaporin-11 containing a divergent NPA motif has normal water channel activity.

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.

Alanine↗
PubMed2006

Neurosteroid pregnenolone sulfate enhances glutamatergic synaptic transmission by facilitating presynaptic calcium currents at the calyx of Held of immature rats.

Pregnenolone sulfate (PREGS) is an endogenous neurosteroid widely released from neurons in the brain, and is thought to play a memory-enhancing role. At excitatory synapses PREGS facilitates transmitter release, but the underlying mechanism is not known. We addressed this issue at the calyx of Held in rat brainstem slices, where direct whole-cell recordings from giant nerve terminals are feasible. PREGS potentiated nerve-evoked excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs, suggesting that its site of action is presynaptic. In whole-cell recordings from calyceal nerve terminals, PREGS facilitated Ca2+ currents, by accelerating their activation kinetics and shifting the half-activation voltage toward negative potentials. PREGS had no effect on presynaptic K+ currents, resting conductance or action potential waveforms. In simultaneous pre- and postsynaptic recordings, PREGS did not change the relationship between presynaptic Ca2+ influx and EPSCs, suggesting that exocytotic machinery downstream of Ca2+ influx is not involved in its effect. PREGS facilitated Ba2+ currents recorded from nerve terminals and also from HEK 293 cells expressed with recombinant N- or P/Q-type Ca2+ channels, suggesting that PREGS-induced facilitation of voltage-gated Ca2+ channels (VGCCs) is neither Ca2+ dependent nor VGCC-type specific. The PREGS-induced VGCC facilitation was blocked by the PREGS scavenger (2-hydroxypropyl)-beta-cyclodextrin applied from outside, but not from inside, of nerve terminals. We conclude that PREGS facilitates VGCCs in presynaptic terminals by acting from outside, thereby enhancing transmitter release. We propose that PREGS may directly modulate VGCCs acting on their extracellular domain.

Animals↗
PubMed2006

Structural basis for detoxification and oxidative stress protection in membranes.

Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 A resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

Amino Acid Sequence↗
PubMed2006

Essential contribution of the ligand-binding beta B/beta C loop of PDZ1 and PDZ2 in the regulation of postsynaptic clustering, scaffolding, and localization of postsynaptic density-95.

Postsynaptic density-95 (PSD-95), a PSD-95/Discs large/zona occludens-1 (PDZ) domain-containing scaffold protein, clusters many signaling molecules near NMDA-type glutamate receptors in the postsynaptic densities. Although the synaptic localization of PSD-95 requires palmitoylation of two cysteines at the N terminus and the presence of at least one PDZ domain, how the clustering of PSD-95 is initiated and regulated remains essentially unknown. To address this issue, we examined PSD-95 clustering in primary cultured hippocampal neurons expressing full-length PSD-95 mutant proteins lacking the ligand-binding ability of PDZ1, PDZ2, and/or PDZ3. The formation of either excitatory or inhibitory synapses was unaffected. Combinations of individual mutations, however, significantly reduced the PSD-95 clustering index, in an approximately additive manner. The sensitivity to 2-bromo-palmitate and latrunculin A, reagents known to affect PSD-95 turnover, was also augmented. Furthermore, the synaptic recruitment of a PSD-95 ligand, synaptic GTPase-activating protein (synGAP), was significantly impaired, whereas the clustering of other scaffolding proteins, such as Homer 1c, Shank/Synamon, and PSD-93/Chapsin-110 was spared. Intriguingly, overexpression of the PSD-95 PDZ1/2/3 mutants caused the PSD-95 clusters to localize away from the dendritic shaft, resulting in the formation of elongated spines, in an inverse correlation with the overall PDZ-ligand affinity. Expression of a mutant synGAP lacking the PDZ-binding motif replicated both the clustering and spine morphology phenotypes. In conclusion, the ligand-binding affinity of the PDZ domains of PSD-95, contributed in part via its interaction with the C-terminal end of synGAP, plays a critical role in titrating the synaptic clustering of PSD-95 and controlling its tight association with the PSD scaffold, thereby affecting synapse maturation.

Animals↗
PubMed2005

Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase.

H(+)-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V(1) and V(o) domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 microm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23A. The Fourier transforms of the untilted images revealed unit-cell dimensions of a=232A, b=132A, and gamma=90 degrees , and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22(1)2(1) symmetry of the crystals and dimer contacts between the V(1) domains.

Crystallization↗
PubMed2005

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.

Animals↗
PubMed2005

Implications of the aquaporin-4 structure on array formation and cell adhesion.

Aquaporin-4 (AQP4) is the predominant water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder and mesial temporal lobe epilepsy. We determined the AQP4 structure by electron crystallography of double-layered, two-dimensional (2D) crystals. The structure allows us to discuss how the expression ratio between the long and short AQP4 splicing variant can determine the size of in vivo orthogonal arrays. Furthermore, AQP4 contains a short 3(10) helix in an extracellular loop, which mediates weak but specific interactions between AQP4 molecules in adjoining membranes. This finding suggests a previously unexpected role for AQP4 in cell adhesion. This notion was corroborated by expression of AQP4 in L-cells, which resulted in clustering of the cells. Our AQP4 structure thus enables us to propose models for the size regulation of orthogonal arrays and channel-mediated cell adhesion.

Amino Acid Sequence↗
PubMed2005

Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state.

We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.

Cell Line, Tumor↗
PubMed2005

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor.

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.

Amino Acid Sequence↗
PubMed2005

Expression and localization of an exogenous G protein-coupled receptor fused with the rhodopsin C-terminal sequence in the retinal rod cells of knockin mice.

Vertebrate rod cell outer segments are highly differentiated compartments consisting of closely packed disk membranes, in which the photoreceptor rhodopsin is embedded at high density. To explore the unusually efficient mechanism of rhodopsin biosynthesis, folding and transport, we challenged it with the ectopic expression in rod cells of human endothelin receptor subtype B (hET(B)R) fused with the C-terminal 10 residues of rhodopsin, under the control of the mouse opsin promoter/enhancer, by gene targeted replacement (knockin), because the C-terminal eight residues are essential to target rhodopsin to the outer segment. The hET(B)R, a type-I G protein-coupled receptor, was successfully expressed and folded in a functional structure in the rod cells of knockin mice. However, while the mRNA level of hET(B)R was one tenth of that of rhodopsin, the hET(B)R protein level was approximately one-thousandth of the rhodopsin level in heterozygous mice, suggesting an intrinsically distinct efficiency in the production of functional receptor protein. In addition, a substantial fraction of the hET(B)R was successfully transported to the outer segment, suggesting that the addition of the C-terminal sequence of rhodopsin enabled hET(B)R to be translocated to the outer segment.

Animals↗
PubMed2004

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains.

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.

Animals↗
PubMed2004

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique.

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.

Aquaporin 4↗
PubMed2004

Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells.

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor that mediates a variety of signals by binding to vasoconstrictive peptides, endothelins. Monoclonal antibodies were prepared against human ET(B)R using the full-length protein expressed in Sf9 cells. Five typical monoclonal antibodies were characterized further for their recognition. The epitopes for the 2A5, 9A3 and 21A1 antibodies were mapped within the N-terminal extracellular sequences, V71-I85 and E27-Q41, respectively, which differ between the human and mouse ET(B)Rs. All of these antibodies labeled cell surface ET(B)R expressed in COS cells, suggesting that their recognition sites exist in the extracellular domain. In addition, the immobilized antibodies could purify ET(B)R expressed in Sf9 cells to the majority under mild conditions. Thus, immunization with the recombinant full-length membrane protein provides a strategy to produce monoclonal antibodies recognizing the native protein.

Amino Acid Sequence↗
PubMed2003

Structure and gating mechanism of the acetylcholine receptor pore.

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.

Acetylcholine↗
PubMed2003

Structural genomics of membrane proteins.

A program on the structural genomics of membrane proteins has started at the BIRC, AIST, involving other academic institutions and industrial companies. Emphasis is being put on the development of techniques for the structural determination of membrane proteins of biological importance and ligand-receptor interactions by means of electron microscopy, X-ray diffraction, NMR, and computer simulation. Most efforts at the present stage, however, are being directed to finding suitable expression and purification systems and crystallization conditions for such proteins. The program is expected to be linked with the human full-length cDNA project and should lead to medical and industrial uses.

Bacteriorhodopsins↗