Takeshi Sato

Osaka University, Suika, Ōsaka, Japan

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Publications (24)131.96 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Activation of the protein tyrosine kinase receptors requires coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 (FGFR3) to assess how activating mutations in the TM region (G380R and A391E) influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM-JM region functionally bridges the extracellular and intracellular regions for these receptors.
    Biochemistry 07/2014; 53(30). DOI:10.1021/bi500327q · 3.19 Impact Factor
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    ABSTRACT: Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.
    Proceedings of the National Academy of Sciences 01/2013; 110(7). DOI:10.1073/pnas.1211560110 · 9.81 Impact Factor
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    ABSTRACT: The transmembrane (TM) and juxtamembrane (JM) regions of the ErbB family receptor tyrosine kinases connect the extracellular ligand-binding domain to the intracellular kinase domain. Evidence for the role of these regions in the mechanism of receptor dimerization and activation is provided by TM-JM peptides corresponding to the Neu (or rat ErbB2) receptor. Solid-state NMR and fluorescence spectroscopy show that there are tight interactions of the JM sequence with negatively charged lipids, including phosphatidylinositol 4,5-bisphosphate, in TM-JM peptides corresponding to the wild-type receptor sequence. We observe a release of the JM sequence from the negatively charged membrane surface using peptides containing an activating V664E mutation within the TM domain or in peptides engineered to form TM helix dimers with Val664 in the interface. These results provide the basis of a mechanism for coupling ligand binding to kinase activation in the full-length receptor.
    Proceedings of the National Academy of Sciences 01/2013; DOI:10.1073/pnas.1215207110 · 9.81 Impact Factor
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    ABSTRACT: The subunit c-ring of H(+)-ATP synthase (F(o) c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [(13)C, (15)N]-labeled F(o) c from E. coli (EF(o) c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the (13)C and (15)N signals were assigned. The obtained chemical shifts suggested that EF(o) c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EF(o) c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EF(o) c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the (13)C nuclei distance of [3-(13)C]Ala24 and [4-(13)C]Asp61 in the F(o) c-ring did not agree with the model structures proposed for the EF(o) c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EF(o) c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EF(o) c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.
    Journal of Biomolecular NMR 09/2010; 48(1):1-11. DOI:10.1007/s10858-010-9432-x · 3.31 Impact Factor
  • Takeshi Sato, Saburo Aimoto
    Seikagaku. The Journal of Japanese Biochemical Society 06/2010; 82(6):498-504. · 0.04 Impact Factor
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    ABSTRACT: The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.
    Nature Structural & Molecular Biology 04/2010; 17(5):561-7. DOI:10.1038/nsmb.1799 · 11.63 Impact Factor
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    ABSTRACT: An efficient method of peptide thioester synthesis is described. The reaction is based on an N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) auxiliary-assisted N-S acyl shift reaction after assembling a peptide chain by Fmoc-solid phase peptide synthesis. The Dmmb-assisted N-S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S-peptide with sodium 2-mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on-resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41-mer peptide thioester, a phosphorylated peptide thioester and a 33-mer peptide thioester containing a trimethylated lysine residue.
    Journal of Peptide Science 11/2009; 15(11):731-7. DOI:10.1002/psc.1164 · 1.86 Impact Factor
  • Alzheimer's and Dementia 07/2009; 5(4). DOI:10.1016/j.jalz.2009.05.223 · 17.47 Impact Factor
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    ABSTRACT: Significant advances have been achieved in the fields of peptide/protein synthesis, permitting the preparation of large, complex molecules. Shortcomings, however, continue to exist in the area of peptide purification. This paper details some studies we undertook to develop a new strategy for peptide purification based on a reactivity of alpha-ketoacyl groups in peptides. The alpha-ketoacyl peptide was generated from N(epsilon)-acyl-lysyl-peptide in the solid phase via a transamination reaction using glyoxylic acid and nickel(II) ion. Cleavage of the alpha-ketoacyl group with o-phenylenediamine gave the target peptide in an acceptable yield and purity. We first carried out a careful step-by-step optimization of the purification conditions using a model peptide. The strategy was then used in the purification of a transmembrane peptide that could not be effectively purified using a conventional RP-HPLC system due to the strong hydrophobicity of the peptide and its high tendency to aggregate.
    Journal of Peptide Science 05/2009; 15(5):369-76. DOI:10.1002/psc.1127 · 1.86 Impact Factor
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    ABSTRACT: The combination of magic angle spinning (MAS) with the high-resolution (1)H NOESY NMR experiment is an established method for measuring through-space (1)H...(1)H dipolar couplings in biological membranes. The segmental motion of the lipid acyl chains along with the overall rotational diffusion of the lipids provides sufficient motion to average the (1)H dipolar interaction to within the range where MAS can be effective. One drawback of the approach is the relatively long NOESY mixing times needed for relaxation processes to generate significant crosspeak intensity. In order to drive magnetization transfer more rapidly, we use solid-state radiofrequency driven dipolar recoupling (RFDR) pulses during the mixing time. We compare the (1)H MAS NOESY experiment with a (1)H MAS RFDR experiment on dimyristoylphosphocholine, a bilayer-forming lipid and show that the (1)H MAS RFDR experiment provides considerably faster magnetization exchange than the standard (1)H MAS NOESY experiment. We apply the method to model compounds containing basic and aromatic amino acids bound to membrane bilayers to illustrate the ability to locate the position of aromatic groups that have penetrated to below the level of the lipid headgroups.
    Journal of Magnetic Resonance 03/2009; 197(1):77-86. DOI:10.1016/j.jmr.2008.12.009 · 2.32 Impact Factor
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    ABSTRACT: Processing of amyloid precursor protein (APP) by gamma-secretase is the last step in the formation of the Abeta peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we show that the TM domain forms an alpha-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the epsilon-cleavage site. This helix-to-coil transition is required for gamma-secretase processing; mutations that extend the TM alpha-helix inhibit epsilon cleavage, leading to a low production of Abeta peptides and an accumulation of the alpha- and beta-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM alpha-helix.
    Proceedings of the National Academy of Sciences 02/2009; 106(5):1421-6. DOI:10.1073/pnas.0812261106 · 9.81 Impact Factor
  • Biophysical Journal 02/2009; 96(3). DOI:10.1016/j.bpj.2008.12.3806 · 3.83 Impact Factor
  • Alzheimer's and Dementia 07/2008; 4(4). DOI:10.1016/j.jalz.2008.05.1007 · 17.47 Impact Factor
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    ABSTRACT: The beta-amyloid peptide (Abeta) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. Abeta is produced by the sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. Cleavage of APP by gamma-secretase also generates the APP intracellular C-terminal domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three Gly-XXX-Gly (GXXXG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GXXXG motif led to a drastic reduction of Abeta40 and Abeta42 secretion. beta-Cleavage of mutant APP was not inhibited, and reduction of Abeta secretion resulted from inhibition of gamma-cleavage. It was anticipated that decreased gamma-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GXXXG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM alpha-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.
    Journal of Biological Chemistry 04/2008; 283(12):7733-44. DOI:10.1074/jbc.M707142200 · 4.60 Impact Factor
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
    ChemInform 06/2007; 38(24). DOI:10.1002/chin.200724226
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    ABSTRACT: The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.
    Biochemistry 11/2006; 45(42):12704-14. DOI:10.1021/bi061264m · 3.19 Impact Factor
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    Wenyi Zhang, Takeshi Sato, Steven O. Smith
    Progress in Nuclear Magnetic Resonance Spectroscopy 08/2006; 48(4). DOI:10.1016/j.pnmrs.2006.04.002 · 8.71 Impact Factor
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    ABSTRACT: Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.
    Biochemistry 06/2006; 45(17):5503-16. DOI:10.1021/bi052485f · 3.19 Impact Factor
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    ABSTRACT: Soluble oligomers and protofibrils are widely thought to be the toxic forms of the Abeta42 peptide associated with Alzheimer's disease. We have investigated the structure and formation of these assemblies using a new approach in atomic force microscopy (AFM) that yields high-resolution images of hydrated proteins and allows the structure of the smallest molecular weight (MW) oligomers to be observed and characterized. AFM images of monomers, dimers and other low MW oligomers at early incubation times (< 1h) are consistent with a hairpin structure for the monomeric Abeta42 peptide. The low MW oligomers are relatively compact and have significant order. The most constant dimension of these oligomers is their height (approximately 1-3 nm) above the mica surface; their lateral dimensions (width and length) vary between 5 nm and 10nm. Flat nascent protofibrils with lengths of over 40 nm are observed at short incubation times (< or = 3h); their lateral dimensions of 6-8 nm are consistent with a mass-per-length of 9 kDa/nm previously predicted for the elementary fibril subunit. High MW oligomers with lateral dimensions of 15-25 nm and heights ranging from 2-8 nm are common at high concentrations of Abeta. We show that an inhibitor designed to block the sheet-to-sheet packing in Abeta fibrils is able to cap the heights of these oligomers at approximately 4 nm. The observation of fine structure in the high MW oligomers suggests that they are able to nucleate fibril formation. AFM images obtained as a function of incubation time reveal a sequence of assembly from monomers to soluble oligomers and protofibrils.
    Journal of Molecular Biology 05/2006; 358(1):106-19. DOI:10.1016/j.jmb.2006.01.042 · 3.96 Impact Factor
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    ABSTRACT: Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (delta5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, delta5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. Delta5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the delta5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces.
    Blood 04/2006; 107(5):1864-71. DOI:10.1182/blood-2005-06-2600 · 9.78 Impact Factor

Publication Stats

792 Citations
131.96 Total Impact Points


  • 2002–2014
    • Osaka University
      • Institute for Protein Research
      Suika, Ōsaka, Japan
  • 2006–2009
    • Stony Brook University
      • Department of Biochemistry and Cell Biology
      Stony Brook, NY, United States
    • Yale University
      • Department of Molecular Biophysics and Biochemistry
      New Haven, Connecticut, United States