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111.
Shengchen Tao Daiju Yamazaki Shinji Komazaki Chengzhu Zhao Tsunaki Iida Sho Kakizawa Yuji Imaizumi Hiroshi Takeshima 《The Journal of biological chemistry》2013,288(22):15581-15589
The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca2+ release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca2+ release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca2+ sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca2+ transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca2+ sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca2+ sparks were facilitated and cell surface Ca2+-dependent K+ channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca2+ channels were inactivated, and thus, the resting intracellular Ca2+ levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca2+ signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level. 相似文献
112.
Atsushi Mukaiyama Takashi Nakamura Koki Makabe Kosuke Maki Yuji Goto Kunihiro Kuwajima 《Journal of molecular biology》2013,425(2):257-272
The kinetic folding of β2-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C). 相似文献
113.
Hatsumi Yoshiki Matomo Nishio Yuji Ikegaya Naoya Takahashi Norio Matsuki Yasuhisa Fujibayashi Yoshiharu Yonekura Toshihiko Momiyama Ikunobu Muramatsu 《Journal of neurochemistry》2013,126(3):360-371
Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system. 相似文献
114.
Atsushi Mukaiyama Takashi Nakamura Koki Makabe Kosuke Maki Yuji Goto Kunihiro Kuwajima 《Journal of molecular biology》2013,425(2):273-291
The acid transition of β2-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 °C). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pKa value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state → MG → burst-phase intermediate → native state. 相似文献
115.
Yuji Inoue Ritsuko Kubota-Koketsu Akifumi Yamashita Mitsuhiro Nishimura Shoji Ideno Ken-ichiro Ono Yoshinobu Okuno Kazuyoshi Ikuta 《The Journal of biological chemistry》2013,288(7):4981-4990
The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains. 相似文献
116.
Yoshiaki Suzuki Hisao Yamamura Susumu Ohya Yuji Imaizumi 《The Journal of biological chemistry》2013,288(51):36750-36761
L-type voltage-dependent Ca2+ channels (LVDCC) and large conductance Ca2+-activated K+ channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca2+ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca2+ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K+ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca2+ dynamics including the negative feedback, to control the arterial excitability and contractility. 相似文献
117.
Yuji Amano Kazuhisa Yoshino Katsuhiko Kojima Toshikazu Takeshita 《Biochemical and biophysical research communications》2013
Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation. 相似文献
118.
Oxidative damage associated with obesity is prevented by overexpression of CuZn- or Mn-superoxide dismutase 总被引:1,自引:0,他引:1
Yuhong Liu Wenbo Qi Arlan Richardson Holly Van Remmen Yuji Ikeno Adam B. Salmon 《Biochemical and biophysical research communications》2013
The development of insulin resistance is the primary step in the etiology of type 2 diabetes mellitus. There are several risk factors associated with insulin resistance, yet the basic biological mechanisms that promote its development are still unclear. There is growing literature that suggests mitochondrial dysfunction and/or oxidative stress play prominent roles in defects in glucose metabolism. Here, we tested whether increased expression of CuZn-superoxide dismutase (Sod1) or Mn-superoxide dismutase (Sod2) prevented obesity-induced changes in oxidative stress and metabolism. Both Sod1 and Sod2 overexpressing mice were protected from high fat diet-induced glucose intolerance. Lipid oxidation (F2-isoprostanes) was significantly increased in muscle and adipose with high fat feeding. Mice with increased expression of either Sod1 or Sod2 showed a significant reduction in this oxidative damage. Surprisingly, mitochondria from the muscle of high fat diet-fed mice showed no significant alteration in function. Together, our data suggest that targeting reduced oxidative damage in general may be a more applicable therapeutic target to prevent insulin resistance than is improving mitochondrial function. 相似文献
119.
Of the 19 strains of Rhizopus delemar deposited as Rhizopus oryzae, seven of them, NBRC 4726, NBRC 4734, NBRC 4746, NBRC 4754, NBRC 4773, NBRC 4775, and NBRC 4801, completely hydrolyzed exogenous sucrose and fructooligosaccharides. The sucrose-hydrolyzing enzyme was purified from the culture filtrate of R. delemar NBRC 4754 and classified to β-fructofuranosidase, similar to that of Amylomyces rouxii CBS 438.76. Fragments including β-fructofuranosidase genes (sucA) of seven strains of R. delemar and A. rouxii CBS 438.76 were amplified and sequenced by PCR with degenerated primers synthesized on the basis of the internal amino acid sequences of purified enzymes and successive inverse PCR. Nucleotide sequences of the obtained fragments revealed that open reading frames of 1,569 bp have no intron and encode 522 amino acids. The presumed proteins contained the typical domain of the glycoside hydrolase 32 family, including β-fructofuranosidase, inulinase, levanase, and fructosyltransferases. Amino acid sequences of SucA proteins from the seven strains of R. delemar were identical and showed 90.0 % identity with those of A. rouxii CBS 438.76. A dendrogram constructed from these amino acid sequences showed that SucA proteins are more closely related to yeast β-fructofuranosidases than to other fungal enzymes. 相似文献
120.
Yuusuke Tamura Kyouhei Hayashi Naoki Omori Yuji Nishiura Kana Watanabe Nobuyuki Tanaka Masahiko Fujioka Naoki Kouyama Akira Yukimasa Yukari Tanaka Takeshi Chiba Hideki Tanioka Hirohide Nambu Hideo Yukioka Hiroki Sato Takayuki Okuno 《Bioorganic & medicinal chemistry letters》2013,23(1):90-95
Optimization of HTS hit 1 for NPY Y5 receptor binding affinity, CYP450 inhibition, solubility and metabolic stability led to the identification of some orally available oxygen-linker derivatives for in vivo study. Among them, derivative 4i inhibited food intake induced by the NPY Y5 selective agonist, and chronic oral administration of 4i in DIO mice caused a dose-dependent reduction of body weight gain. 相似文献