Unraveling molecular mechanism of cellular homeostasis
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Academic Paper (Judged Full Paper):
1.
Sachiho Taniguchi, Yuji Ono, Yukako Doi, Shogo Taniguchi, Yuta Matsuura, Ayuka Iwasaki, Noriaki Hirata, Ryosuke Fukuda, Keitaro Inoue, Miho Yamaguchi, Anju Tashiro, Daichi Egami, Shunsuke Aoki, Yasumitsu Kondoh, Kaori Honda, Hiroyuki Osada, Hiroyuki Kumeta, Tomohide Saio and Tsukasa Okiyoneda : Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis, Biochemical Pharmacology, Vol.215, 115730, 2023.
(Summary)
The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.
A transcriptional regulatory system called heat shock response (HSR) has been developed in eukaryotic cells to maintain proteome homeostasis under various stresses. Heat shock factor-1 (Hsf1) plays a central role in HSR, mainly by upregulating molecular chaperones as a transcription factor. Hsf1 forms a complex with chaperones and exists as a monomer in the resting state under normal conditions. However, upon heat shock, Hsf1 is activated by oligomerization. Thus, oligomerization of Hsf1 is considered an important step in HSR. However, the lack of information about Hsf1 monomer structure in the resting state, as well as the structural change via oligomerization at heat response, impeded the understanding of the thermosensing mechanism through oligomerization. In this study, we applied solution biophysical methods, including fluorescence spectroscopy, nuclear magnetic resonance, and circular dichroism spectroscopy, to investigate the heat-induced conformational transition mechanism of Hsf1 leading to oligomerization. Our study showed that Hsf1 forms an inactive closed conformation mediated by intramolecular contact between leucine zippers (LZs), in which the intermolecular contact between the LZs for oligomerization is prevented. As the temperature increases, Hsf1 changes to an open conformation, where the intramolecular LZ interaction is dissolved so that the LZs can form intermolecular contacts to form oligomers in the active form. Furthermore, since the interaction sites with molecular chaperones and nuclear transporters are also expected to be exposed in the open conformation, the conformational change to the open state can lead to understanding the regulation of Hsf1-mediated stress response through interaction with multiple cellular components.
Masato Miyake, Mitsuaki Sobajima, Kiyoe Kurahashi, Akira Shigenaga, Masaya Denda, Akira Otaka, Tomohide Saio, Naoki Sakane, Hidetaka Kosako and Seiichi Oyadomari : Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells., Cell Chemical Biology, Vol.29, No.6, 996-1009.e9, 2022.
(Summary)
Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function.
Motonori Matsusaki, Okada Rina, Tanikawa Yuya, Shingo Kanemura, Ito Dai, Lin Yuxi, Watabe Mai, Yamaguchi Hiroshi, Tomohide Saio, Lee Young-Ho, Inaba Kenji and Okumura Masaki : Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding, Biology, Vol.10, No.11, 1112, 2021.
(Summary)
The physiological functions of proteins are destined by their unique three-dimensional structures. Almost all biological kingdoms share conserved disulfide-catalysts and chaperone networks that assist in correct protein folding and prevent aggregation. Disruption of these networks is implicated in pathogenesis, including neurodegenerative disease. In the mammalian endoplasmic reticulum (ER), more than 20 members of the protein disulfide isomerase family (PDIs) are believed to cooperate in the client folding pathway, but it remains unclear whether complex formation among PDIs via non-covalent interaction is involved in regulating their enzymatic and chaperone functions. Herein, we report novel functional hetero complexes between PDIs that promote oxidative folding and inhibit aggregation along client folding. The findings provide insight into the physiological significance of disulfide-catalyst and chaperone networks and clues for understanding pathogenesis associated with disruption of the networks.
(Keyword)
protein disulfide isomerase family / disulfide bond / endoplasmic reticulum / oxidative folding / molecular chaperone / protein-protein interaction
Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment.
Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
(Keyword)
Active Transport, Cell Nucleus / Binding Sites / C9orf72 Protein / Cloning, Molecular / DNA-Binding Proteins / Escherichia coli / Gene Expression / Genetic Vectors / HeLa Cells / Humans / Models, Molecular / Nuclear Localization Signals / Peptides / Phase Transition / Protein Binding / Protein Conformation / Protein Interaction Domains and Motifs / RNA-Binding Protein FUS / Recombinant Proteins / beta Karyopherins
Hiroshi Nakagawa, Tomohide Saio, Michihiro Nagao, Rintaro Inoue, Masaaki Sugiyama, Satoshi Ajito, Taiki Tominaga and Yukinobu Kawakita : Conformational dynamics of a multidomain protein by neutron scattering and computational analysis., Biophysical Journal, Vol.120, No.16, 3341-3354, 2021.
(Summary)
The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.
Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.
(Keyword)
Electron Spin Resonance Spectroscopy / Ligands / Magnetic Resonance Spectroscopy / Molecular Conformation / Proteins
Masahiro Mimura, Shunsuke Tomita, Yoichi Shinkai, Takuya Hosokai, Hiroyuki Kumeta, Tomohide Saio, Kentaro Shiraki and Ryoji Kurita : Quadruplex Folding Promotes the Condensation of Linker Histones and DNAs via Liquid-Liquid Phase Separation., Journal of the American Chemical Society, Vol.143, No.26, 9849-9857, 2021.
(Summary)
Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that, in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with a high charge density. Given that DNA quadruplex structures are well-documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.
Masaki Okumura, Shingo Kanemura, Motonori Matsusaki, Misaki Kinoshita, Tomohide Saio, Dai Ito, Chihiro Hirayama, Hiroyuki Kumeta, Mai Watabe, Yuta Amagai, Young-Ho Lee, Shuji Akiyama and Kenji Inaba : A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization., Structure, Vol.29, No.12, 1357-1370.E6, 2021.
(Summary)
P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on 30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.
Aya Okuda, Rintaro Inoue, Ken Morishima, Tomohide Saio, Yasuhiro Yunoki, Maho Yagi-Utsumi, Hirokazu Yagi, Masahiro Shimizu, Nobuhiro Sato, Reiko Urade, Koichi Kato and Masaaki Sugiyama : Deuteration Aiming for Neutron Scattering., Biophysics and Physicobiology, Vol.18, 16-27, 2021.
(Summary)
The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.
Kamran Rizzolo, Hsiung Angela Yeou Yu, Adedeji Ologbenla, Rang Sa Kim, Haojie Zhu, Koichiro Ishimori, Guillaume Thibault, Elisa Leung, Wen Yi Zhang, Mona Teng, Marta Haniszewski, Noha Miah, Sadhna Phanse, Zoran Minic, Sukyeong Lee, Diaz Julio Caballero, Mohan Babu, F Francis T Tsai, Tomohide Saio and A Walid Houry : Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex., Nature Communications, Vol.12, No.1, 2021.
(Summary)
A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
Rintaro Inoue, Takashi Oda, Hiroshi Nakagawa, Taiki Tominaga, Tomohide Saio, Yukinobu Kawakita, Masahiro Shimizu, Aya Okuda, Ken Morishima, Nobuhiro Sato, Reiko Urade, Mamoru Sato and Masaaki Sugiyama : Dynamics of proteins with different molecular structures under solution condition., Scientific Reports, Vol.10, No.1, 2020.
(Summary)
Incoherent quasielastic neutron scattering (iQENS) is a fascinating technique for investigating the internal dynamics of protein. However, low flux of neutron beam, low signal to noise ratio of QENS spectrometers and unavailability of well-established analyzing method have been obstacles for studying internal dynamics under physiological condition (in solution). The recent progress of neutron source and spectrometer provide the fine iQENS profile with high statistics and as well the progress of computational technique enable us to quantitatively reveal the internal dynamic from the obtained iQENS profile. The internal dynamics of two proteins, globular domain protein (GDP) and intrinsically disordered protein (IDP) in solution, were measured with the state-of-the art QENS spectrometer and then revealed with the newly developed analyzing method. It was clarified that the average relaxation rate of IDP was larger than that of GDP and the fraction of mobile H atoms of IDP was also much higher than that of GDP. Combined with the structural analysis and the calculation of solvent accessible surface area of amino acid residue, it was concluded that the internal dynamics were related to the highly solvent exposed amino acid residues depending upon protein's structure.
(Keyword)
Amino Acids / Intrinsically Disordered Proteins / Molecular Dynamics Simulation / Molecular Structure / Protein Domains / Protein Folding / Protein Structure, Tertiary / Solutions / Solvents / Spectrum Analysis
Yuya Taguchi, Tomohide Saio and Daisuke Kohda : Distance Distribution between Two Iodine Atoms Derived from Small-Angle X-ray Scattering Interferometry for Analyzing a Conformational Ensemble of Heavy Atom-Labeled Small Molecules., The Journal of Physical Chemistry Letters, Vol.11, No.14, 5451-5456, 2020.
(Summary)
To obtain unbiased information about the dynamic conformational ensemble of a molecule in solution, one promising approach is small-angle X-ray scattering (SAXS). Conventionally, SAXS data are converted to a pair distribution function, which describes the distance distribution between all pairs of atoms within a molecule. If two strong X-ray scatterers are introduced and the background contributions from the other atoms are suppressed, then the distance distribution between the two scatterers provides spatial information about a flexible molecule. Gold nanocrystals can provide such information for distances of >50 Å. Here, we synthesized a chemical compound containing two iodine atoms attached to the ends of a flexible polyethylene glycol chain and used the relevant singly labeled and unlabeled compounds to suppress the background contribution. This is a feasibility demonstration to prove that the distance distribution in the range of 10-30 Å can be experimentally accessed by SAXS.
Masaki Okumura, Kentaro Noi, Shingo Kanemura, Misaki Kinoshita, Tomohide Saio, Yuichi Inoue, Takaaki Hikima, Shuji Akiyama, Teru Ogura and Kenji Inaba : Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding., Nature Chemical Biology, Vol.15, No.5, 499-509, 2019.
(Summary)
Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.
(Keyword)
Biocatalysis / endoplasmic reticulum / Humans / Mutation / oxidation and reduction / Protein Conformation / Protein Disulfide-Isomerases / Protein Folding / Substrate Specificity
Soichiro Kawagoe, Hiroshi Nakagawa, Hiroyuki Kumeta, Koichiro Ishimori and Tomohide Saio : isomerization of unfolded proteins catalyzed by the trigger factor chaperone., The Journal of Biological Chemistry, Vol.293, No.39, 15095-15106, 2018.
(Summary)
to form an intermolecular hydrogen bond with the carbonyl oxygen of the amino acid residue preceding the proline residue at the transition state, which presumably stabilizes the transition state and thus accelerates the isomerization. The importance of such intermolecular hydrogen-bond formation during the catalysis was further corroborated by the activity assay and NMR relaxation analysis.
Tomohide Saio, Soichiro Kawagoe, Koichiro Ishimori and G Charalampos Kalodimos : Oligomerization of a molecular chaperone modulates its activity., eLife, Vol.7, 2018.
(Summary)
Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell.
(Keyword)
Binding Sites / Escherichia coli / Escherichia coli Proteins / Molecular Chaperones / Peptidylprolyl Isomerase / Protein Binding / Protein Conformation / Protein Folding / Protein Multimerization
Wataru Sato, Takeshi Uchida, Tomohide Saio and Koichiro Ishimori : Polyethylene glycol promotes autoxidation of cytochrome c., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1862, No.6, 1339-1349, 2018.
(Summary)
Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.
(Keyword)
Cytochromes c / Electron Transport / Heme / Humans / oxidation and reduction / Oxygen / Polyethylene Glycols / Protein Conformation
Takahiro Muraoka, Tomohide Saio and Masaki Okumura : Biophysical elucidation of neural network and chemical regeneration of neural tissue., Biophysics and Physicobiology, Vol.19, 2022.
Soichiro Kawagoe, Koichiro Ishimori and Tomohide Saio : Structural and Kinetic Views of Molecular Chaperones in Multidomain Protein Folding., International Journal of Molecular Sciences, Vol.23, No.5, Feb. 2022.
(Summary)
Despite recent developments in protein structure prediction, the process of the structure formation, folding, remains poorly understood. Notably, folding of multidomain proteins, which involves multiple steps of segmental folding, is one of the biggest questions in protein science. Multidomain protein folding often requires the assistance of molecular chaperones. Molecular chaperones promote or delay the folding of the client protein, but the detailed mechanisms are still unclear. This review summarizes the findings of biophysical and structural studies on the mechanism of multidomain protein folding mediated by molecular chaperones and explains how molecular chaperones recognize the client proteins and alter their folding properties. Furthermore, we introduce several recent studies that describe the concept of kinetics-activity relationships to explain the mechanism of functional diversity of molecular chaperones.
(Keyword)
Humans / Kinetics / Molecular Chaperones / Protein Folding
Kawagoe Soichiro, Mori Eiichiro and Tomohide Saio : Regulation of Liquid-Liquid Phase Separation by Molecular Chaperones, Thermal Medicine, Vol.37, No.2, 31-44, Aug. 2021.
Takuya Yoshizawa, Ryu-Suke Nozawa, Z Tony Jia, Tomohide Saio and Eiichiro Mori : Biological phase separation: cell biology meets biophysics., Biophysical Reviews, Vol.12, No.2, 519-539, Mar. 2020.
(Summary)
Progress in development of biophysical analytic approaches has recently crossed paths with macromolecule condensates in cells. These cell condensates, typically termed liquid-like droplets, are formed by liquid-liquid phase separation (LLPS). More and more cell biologists now recognize that many of the membrane-less organelles observed in cells are formed by LLPS caused by interactions between proteins and nucleic acids. However, the detailed biophysical processes within the cell that lead to these assemblies remain largely unexplored. In this review, we evaluate recent discoveries related to biological phase separation including stress granule formation, chromatin regulation, and processes in the origin and evolution of life. We also discuss the potential issues and technical advancements required to properly study biological phase separation.
<p>Neutron has distinct features as a scattering probe to analyze structure and dynamics of biological macromolecules. The theme of this review is to try to describe how we did/do utilize them. And "How we should utilize them more effectively in the trend of integrative structural biology?" with solution scattering.</p>
Tomohide Saio and Koichiro Ishimori : Accelerating structural life science by paramagnetic lanthanide probe methods., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1864, No.2, 129332, Mar. 2019.
(Summary)
We describe the recent progress of structural analysis methods exploiting paramagnetic lanthanide ions. In NMR spectroscopy, the paramagnetic effects induced by the trivalent lanthanide ions provide long-range (~40 Å) distance and angular information that can be exploited in protein structure determination, ligand screening, structure-based resonance assignment, and in-cell observation. The paramagnetic lanthanide ions can also be utilized in EPR spectroscopy, providing nanometer-scale distance measurement. These applications of the paramagnetic lanthanide probe are becoming more widespread by the use of a variety of lanthanide binding tags. Here, we introduce the basics of paramagnetic effects, several examples of lanthanide tags, and recent applications of paramagnetic lanthanide ions in NMR and EPR spectroscopy. Collectively, we show how the paramagnetic lanthanide probe accelerates research in protein science and drug design, and consequently life science.
(Keyword)
Cysteine / Edetic Acid / Electron Spin Resonance Spectroscopy / Escherichia coli / Lanthanoid Series Elements / Ligands / Models, Molecular / Nuclear Magnetic Resonance, Biomolecular / Peptides / Protein Binding / Protein Conformation / Proteins
Tomohide Saio : Conformational distribution of a multi-domain protein enzyme investigated by paramagnetic NMR and ESR, 3rd India-Japan NMR WORK SHOP, Online, Feb. 2023.
2.
Tomohide Saio : NMR investigation of the regulators in protein folding and assembly, ZOOMinar on Molecular Bases of Proteinopathies, Online, Feb. 2023.
3.
Tomohide Saio : Structural and kinetic views of regulators for protein folding and assembly, International Symposium: Protein Folding, Aggregation, Misfolding Disease, and Disease Crosstalk, Online, Sep. 2022.
4.
Tomohide Saio : Conformational Variation of a Multi-Domain Protein Enzyme Investigated by Paramagnetic Lanthanide Probe, ISMAR-APNMR2021, Aug. 2021.
5.
Tomohide Saio, Hiroshi Nakagawa, Soya Hiramatsu, Mizue Asada, Honoka Kawamukai, Toshikazu Nakamura and Koichiro Ishimori : Application of a lanthanide tag for evaluation of conformational states of a multidomain protein, IUCr 2021 - XXV General Assembly and Congress of the International Union of Crystallography, Aug. 2021.
6.
Tomohide Saio : Mechanistic insights into a molecular chaperone in protein folding and degradation, 2021 virtual Cold Spring Harbor Asia Conference, Mar. 2021.
7.
Tomohide Saio : NMR investigation of molecular chaperones manipulating protein folding, Pacifichem 2021, 2021.
8.
Tomohide Saio : Structural and kinetic insights into a molecular chaperone for protein folding, translocation, and degradation, Pacifichem 2021, 2021.
9.
Kawamukai Honoka, Ishimori Koichiro and Tomohide Saio : ALS-associated factor PRn inhibit the function of KapB2, The 16th International Symposium of the Institute Network for Biomedical Sciences & KEY FORUM 2021 International Symposium, 2021.
10.
Tomohide Saio : Mechanistic insight into chaperone-mediated protein homeostasis, The 16th International Symposium of the Institute Network for Biomedical Sciences & KEY FORUM 2021 International Symposium, 2021.
11.
Tomohide Saio : On and off between molecular chaperones and clients: Appropriate distance and timing for protein folding, 21st Hokudai-RIES International Symposium, Dec. 2020.
Proceeding of Domestic Conference:
1.
Tomohide Saio : ランタノイドタグを用いた蛋白質の動的構造解析, 日本化学会第103春季年会(2023), Mar. 2023.
2.
Tomohide Saio : 常磁性プローブを用いたマルチドメインタンパク質の構造解析, 蛋白研セミナー 基礎から学ぶ最新NMR解析法 第6回ワークショップ 統合型構造生物学研究, Mar. 2023.
3.
Tomohide Saio : Structural insight into regulation and dysregulation of protein assembly and folding, The 1st IMEG Meeting of The Research Center for High Depth Omics, Jan. 2023.
4.
Tomohide Saio : シャペロンから解き明かすタンパク質フォールディングと集合の分子メカニズム, 第18回Organelle zone seminar, Dec. 2022.
Stratification of Protein Aggregation States based on Pattern Recognition for Screening Factors Controlling Phase Separation (Project/Area Number: 23H01995 )
Mechanism of chaperone activity modulation through promiscuous recognitions (Project/Area Number: 22H02560 )
Unraveling the mechanism of kinetic regulation in biomolecular folding (Project/Area Number: 21H05094 )
Organization of kinetics-driven supramolecular chemistry (Project/Area Number: 21H05093 )
Structural basis for recognition and regulation of protein folding intermediate by molecular chaperones in the endoplasmic reticulum (Project/Area Number: 20KK0156 )
Regulatory mechanism of biological phase separation (Project/Area Number: 20H03199 )
Dynamic Structural Characterization of Electron Transfer Complex in Respiratory Chain of Mitochondria and Its Electron Transfer Regulation Mechanism (Project/Area Number: 16H04173 )
Analysis of respiratory electron transport chain using the paramagnetic probes (Project/Area Number: 15K20829 )
Functional and Structural Characterization of Electron Transfer Reactions in Mitochondria Respiratory Chain Utilizing Nanodisc (Project/Area Number: 25288072 )