Publications by authors named "Hideki Taguchi"

86 Publications

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli.

FEBS Lett 2021 Jun 14. Epub 2021 Jun 14.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.

Acetate overflow refers to the metabolism by which a large part of carbon incorporated as glucose into Escherichia coli cells is catabolized and excreted as acetate into the medium. We previously found that mutants for the acetate overflow pathway enzymes phosphoacetyltransferase (Pta) and acetate kinase (AckA) showed significant diauxic growth after glucose depletion in E. coli. Here, we analyzed the underlying mechanism in the pta mutant. Proteomic and other analyses revealed an increase in pyruvate dehydrogenase complex subunits and a decrease in glyoxylate shunt enzymes, which resulted from pyruvate accumulation. Since restoration of these enzyme levels by overexpressing PdhR (pyruvate-sensing transcription factor) or deleting iclR (gene encoding a pyruvate- and glyoxylate-sensing transcription factor) alleviated the growth lag of the pta mutant after glucose depletion, these changes were considered as the reason for the phenotype. Given the evidence for decreased coenzyme A (HS-CoA) levels in the pta mutant, the growth inhibition after glucose depletion was partly explained by limited availability of HS-CoA in the cell. The findings provide insights into the role of acetate overflow in metabolic regulation, which may be useful for biotechnological applications.
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http://dx.doi.org/10.1002/1873-3468.14151DOI Listing
June 2021

Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.

J Am Chem Soc 2021 May 27;143(20):7726-7731. Epub 2021 Apr 27.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (O) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of O and a technique to localize the O generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that O-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.
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http://dx.doi.org/10.1021/jacs.1c01626DOI Listing
May 2021

Novel self-regulation strategy of a small heat shock protein for prodigious and rapid expression on demand.

Curr Genet 2021 Apr 10. Epub 2021 Apr 10.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, S2-19, Nagatsuta 4259, Midori-ku, Yokohama, 226-8503, Japan.

In this mini-review, we summarize the known and novel regulation mechanisms of small heat shock proteins (sHsps). sHsps belong to a well-conserved family of ATP-independent oligomeric chaperones that protect denatured proteins from forming irreversible aggregates by co-aggregation. The functions of sHsps as a first line of defense against acute stresses require the high abundance of sHsps on demand. The heat stress-induced expression of IbpA, one of the sHsps in Escherichia coli, is regulated by σ, an RNA polymerase subunit, and the thermoresponsive mRNA structures in the 5' untranslated region, called RNA thermometers. In addition to the known mechanisms, a recent study has revealed unexpected processes by which the oligomeric IbpA self-represses the ibpA translation via the direct binding of IbpA to its own mRNA, and mediates the mRNA degradation. In summary, the role of IbpA as an aggregation-sensor, combined with other mechanisms, tightly regulates the expression level of IbpA, thus enabling the sHsp to function as a "sequestrase" upon acute aggregation stress, and provides new insights into the mechanisms of other sHsps in both bacteria and eukaryotes.
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http://dx.doi.org/10.1007/s00294-021-01185-0DOI Listing
April 2021

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

iScience 2021 Feb 13;24(2):102059. Epub 2021 Jan 13.

Department of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.
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http://dx.doi.org/10.1016/j.isci.2021.102059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848650PMC
February 2021

Thioredoxin pathway in anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C.

J Biochem 2021 Feb 4. Epub 2021 Feb 4.

Laboratory for Chemistry and Life Science, Institute for Innovative Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama, 226-8503, Japan.

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and PSI subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity, and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-Cys Prx was down-regulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP, and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark to light transition period.
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http://dx.doi.org/10.1093/jb/mvab014DOI Listing
February 2021

G-quadruplex-proximity protein labeling based on peroxidase activity.

Chem Commun (Camb) 2020 Oct 1;56(78):11641-11644. Epub 2020 Sep 1.

Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, Japan.

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.
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http://dx.doi.org/10.1039/d0cc02571bDOI Listing
October 2020

Escherichia coli small heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at posttranscriptional levels.

Mol Microbiol 2021 01 14;115(1):142-156. Epub 2020 Oct 14.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the σ heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsps expression. Oligomeric IbpA self-represses the ibpA/ibpB translation, and mediates its own mRNA degradation, but the self-repression is relieved by sequestration of IbpA into the protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.
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http://dx.doi.org/10.1111/mmi.14606DOI Listing
January 2021

Molecularly Engineered "Janus GroEL": Application to Supramolecular Copolymerization with a Higher Level of Sequence Control.

J Am Chem Soc 2020 08 27;142(31):13310-13315. Epub 2020 Jul 27.

Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Herein we report the synthesis and isolation of a shape-persistent Janus protein nanoparticle derived from the biomolecular machine chaperonin GroEL () and its application to DNA-mediated ternary supramolecular copolymerization. To synthesize with two different DNA strands and at its opposite apical domains, we utilized the unique biological property of GroEL, i.e., Mg/ATP-mediated ring exchange between and with their hollow cylindrical double-decker architectures. This exchange event was reported more than 24 years ago but has never been utilized for molecular engineering of GroEL. We leveraged DNA nanotechnology to purely isolate Janus and succeeded in its precision ternary supramolecular copolymerization with two DNA comonomers, and , that are partially complementary to and in , respectively, and programmed to self-dimerize on the other side. Transmission electron microscopy allowed us to confirm the formation of the expected dual-periodic copolymer sequence -()- in the form of a laterally connected lamellar assembly rather than a single-chain copolymer.
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http://dx.doi.org/10.1021/jacs.0c05937DOI Listing
August 2020

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

Proc Natl Acad Sci U S A 2020 04 25;117(14):7831-7836. Epub 2020 Mar 25.

School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, 226-8503 Yokohama, Japan;

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [ ]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.
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http://dx.doi.org/10.1073/pnas.1916452117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149427PMC
April 2020

Nascent SecM chain interacts with outer ribosomal surface to stabilize translation arrest.

Biochem J 2020 01;477(2):557-566

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57-98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57-98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.
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http://dx.doi.org/10.1042/BCJ20190723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993859PMC
January 2020

Catalyst-proximity protein chemical labelling on affinity beads targeting endogenous lectins.

Chem Commun (Camb) 2019 Oct;55(88):13275-13278

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-13, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.

Magnetic affinity beads functionalized with lactose and ruthenium/dcbpy complexes were developed. Using MAUra, a catalyst-proximity labelling reagent, the catalytic labeling of lactose-binding proteins was achieved with high selectivity on the beads. The first unbiased identification of cellular endogenous lectins bound to lactose (galectin-1 and galectin-3) was achieved with chemical labelling on the affinity beads.
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http://dx.doi.org/10.1039/c9cc05231cDOI Listing
October 2019

Large-scale analysis of diffusional dynamics of proteins in living yeast cells using fluorescence correlation spectroscopy.

Biochem Biophys Res Commun 2019 12 5;520(2):237-242. Epub 2019 Oct 5.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan; Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan. Electronic address:

In the living cells, the majority of proteins does not work alone, but interact with other proteins or other biomolecules to maintain the cellular function, constituting a "protein community". Previous efforts on mass spectroscopy-based protein interaction networks, interactomes, have provided a picture on the protein community. However, these were static information after cells were disrupted. For a better understanding of the protein community in cells, it is important to know the properties of intracellular dynamics and interactions. Since hydrodynamic size and mobility of proteins are related into such properties, direct measurement of diffusional motion of proteins in single living cells will be helpful for uncovering the properties. Here we completed measurement of the diffusion and homo-oligomeric properties of 369 cytoplasmic GFP-fusion proteins in living yeast Saccharomyces cerevisiae cells using fluorescence correlation spectroscopy (FCS). The large-scale analysis showed that the motions of majority of proteins obeyed a two-component (i.e. slow and fast components) diffusion model. Remarkably, both of the two components diffused more slowly than expected monomeric states. In addition, further analysis suggested that more proteins existed as homo-oligomeric states in living cells than previously expected. Our study, which characterizes the dynamics of proteins in living cells on a large-scale, provided a global view on intracellular protein dynamics to understand the protein community.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.066DOI Listing
December 2019

Disruption of the Gene trx-m1 Impedes the Growth of Anabaena sp. PCC 7120 under Nitrogen Starvation.

Plant Cell Physiol 2019 Jul;60(7):1504-1513

Laboratory for Chemistry and Life Science, Institute for Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama, Japan.

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.
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http://dx.doi.org/10.1093/pcp/pcz056DOI Listing
July 2019

Translation-coupled protein folding assay using a protease to monitor the folding status.

Protein Sci 2019 07 3;28(7):1252-1261. Epub 2019 May 3.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated ∼90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.
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http://dx.doi.org/10.1002/pro.3624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567683PMC
July 2019

Proteome Analysis of Phase-Separated Condensed Proteins with Ionic Surfactants Revealed Versatile Formation of Artificial Biomolecular Condensates.

Biomacromolecules 2019 01 21;20(1):539-545. Epub 2018 Dec 21.

Cell Biology Center, Institute of Innovative Research , Tokyo Institute of Technology , 4259 Nagatsuta-Cho, Midori-ku, Yokohama , Kanagawa 226-8503 , Japan.

The formation of highly condensed, native proteins is important for the development of protein-based drugs and materials. In the cell, various types of liquid droplets with broad functions are formed by the spontaneous condensation of protein, as a physiological response. These droplets lack a surrounding membrane but are phase-separated from the water medium. These types of phase-separated states of proteins have potential applications in biotechnology. Recently, we have developed an artificial phase-separated liquid of condensed native proteins, termed a protein condensate (PC), formed by electrostatic complexation with ionic surfactants. Here we report the applicability of PC formation, studied using an E. coli extract as the protein source. The addition of anionic and cationic surfactants at a specific ratio to the E. coli extract resulted in PC formation. A proteome analysis showed that the PC thus formed contained about 600 kinds of proteins, representing 65% of the uniquely detected proteins and confirming the high versatility of PC formation. A statistical analysis revealed that a variety of types of proteins with a wide range of molecular weights and isoelectric points could form PCs.
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http://dx.doi.org/10.1021/acs.biomac.8b01379DOI Listing
January 2019

A LEA model peptide protects the function of a red fluorescent protein in the dry state.

Biochem Biophys Rep 2019 Mar 26;17:27-31. Epub 2018 Nov 26.

Center for Biological Resources and Informatics, Tokyo Institute of Technology, B-62 4259, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide.
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http://dx.doi.org/10.1016/j.bbrep.2018.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6259040PMC
March 2019

Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation.

J Biol Chem 2018 12 16;293(50):19228-19239. Epub 2018 Oct 16.

From the Department of Biology, Faculty of Science and Engineering and

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA), ClpB forms a hexameric ring structure, with each protomer containing two AAA modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from (ClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed ClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.
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http://dx.doi.org/10.1074/jbc.RA118.005496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302173PMC
December 2018

The Absence of Thioredoxin m1 and Thioredoxin C in Anabaena sp. PCC 7120 Leads to Oxidative Stress.

Plant Cell Physiol 2018 Dec;59(12):2432-2441

Laboratory for Chemistry and Life Science, Institute for Innovative Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama, Japan.

Thioredoxin (Trx) family proteins perform redox regulation in cells, and they are involved in several other biological processes (e.g. oxidative stress tolerance). In the filamentous cyanobacterium Anabaena sp. PCC7120 (A. 7120), eight Trx isoforms have been identified via genomic analysis. Among these Trx isoforms, the absence of Trx-m1 and TrxC appears to result in oxidative stress in A. 7120 together with alterations of the thylakoid membrane structure and phycobiliprotein composition. To analyze the physiological changes in these Trx disruptants thoroughly, quantitative proteomics was applied. Certainly, the mutants exhibited similar alterations in the proteome including decreased relative abundance of phycobiliproteins and an increased level of proteins involved in amino acid and carbohydrate metabolism. Nevertheless, the results also indicated that the mutants exhibited changes in the relative abundance of different sets of proteins participating in reactive oxygen species detoxification, such as Fe-SOD in Δtrx-m1 and PrxQ in ΔtrxC, suggesting distinct functions of Trx-m1 and TrxC.
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http://dx.doi.org/10.1093/pcp/pcy163DOI Listing
December 2018

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding.

Sci Rep 2018 01 12;8(1):678. Epub 2018 Jan 12.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.

A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.
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http://dx.doi.org/10.1038/s41598-017-18977-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766493PMC
January 2018

Protein Nanotube Selectively Cleavable with DNA: Supramolecular Polymerization of "DNA-Appended Molecular Chaperones".

J Am Chem Soc 2018 01 13;140(1):26-29. Epub 2017 Dec 13.

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Here, we report molecular chaperone GroELs that carry, at their apical domains, multiple DNA strands (ideally 28 DNA strands in total) with defined oligonucleotide (nt) sequences. This design strategy allows for the preparation of GroEL and GroEL carrying 10-nt DNA strands of 10a and 10b with complementary sequences, respectively, at their apical domains. One-dimensional coassembly of these GroELs is possible to form protein nanotube NT with an anomalous thermodynamic stability due to the exceptionally large multivalency for the coassembly. Likewise, comparably stable nanotube NT was obtained even when the apical-domain DNA strands (15c and 10d) were partially complementary to one another. Nevertheless, in sharp contrast with NT, NT, when incubated with DNA 15d, dissociates rapidly and completely because 15d preferentially hybridizes with the DNA strands of 15c in NT by displacing those of 10d, to afford a mixture of GroEL and GroEL. Even in the presence of NT, 15d cleaved off NT selectively, indicating the potential utility of NTs for targeted delivery.
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http://dx.doi.org/10.1021/jacs.7b09892DOI Listing
January 2018

Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Mol Cell 2017 Nov;68(3):528-539.e5

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan. Electronic address:

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg sensor by harnessing IRD.
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http://dx.doi.org/10.1016/j.molcel.2017.10.020DOI Listing
November 2017

In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile.

Nucleic Acids Res 2017 Nov;45(19):11449-11458

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.
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http://dx.doi.org/10.1093/nar/gkx776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737407PMC
November 2017

Heterogeneous interaction network of yeast prions and remodeling factors detected in live cells.

BMB Rep 2017 Sep;50(9):478-483

Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

Budding yeast has dozens of prions, which are mutually dependent on each other for the de novo prion formation. In addition to the interactions among prions, transmissions of prions are strictly dependent on two chaperone systems: the Hsp104 and the Hsp70/Hsp40 (J-protein) systems, both of which cooperatively remodel the prion aggregates to ensure the multiplication of prion entities. Since it has been postulated that prions and the remodeling factors constitute complex networks in cells, a quantitative approach to describe the interactions in live cells would be required. Here, the researchers applied dual-color fluorescence cross-correlation spectroscopy to investigate the molecular network of interaction in single live cells. The findings demonstrate that yeast prions and remodeling factors constitute a network through heterogeneous protein-protein interactions. [BMB Reports 2017; 50(9): 478-483].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625696PMC
http://dx.doi.org/10.5483/bmbrep.2017.50.9.084DOI Listing
September 2017

Supramolecular Nanotube of Chaperonin GroEL: Length Control for Cellular Uptake Using Single-Ring GroEL Mutant as End-Capper.

J Am Chem Soc 2016 09 25;138(35):11152-5. Epub 2016 Aug 25.

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

How to modulate supramolecular protein nanotubes without sacrificing their thermodynamic stability? This challenging issue emerged with an enhanced reality since our successful development of a protein nanotube of chaperonin GroELMC as a novel ATP-responsive 1D nanocarrier because the nanotube length may potentially affect the cellular uptake efficiency. Herein, we report a molecularly engineered protein end-capper (SRMC) that firmly binds to the nanotube termini since the end-capper originates from GroEL. According to the single-ring mutation of GroEL, we obtained a single-ring version of GroEL bearing cysteine mutations (GroELCys) and modified its 14 apical cysteine residues with merocyanine (MC). Whereas SRMC self-dimerizes upon treatment with Mg(2+), we confirmed that SRMC serves as the efficient end-capper for the Mg(2+)-mediated supramolecular polymerization of GroELMC and allows for modulating the average nanotube length over a wide range from 320 to 40 nm by increasing the feed molar ratio SRMC/GroELMC up to 5.4. We also found that the nanotubes shorter than 100 nm are efficiently taken up into HEP3B cells.
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http://dx.doi.org/10.1021/jacs.6b07925DOI Listing
September 2016

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing.

Proc Natl Acad Sci U S A 2016 Feb 1;113(7):E829-38. Epub 2016 Feb 1.

Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan;

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.
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http://dx.doi.org/10.1073/pnas.1520560113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763732PMC
February 2016

Identification of novel in vivo obligate GroEL/ES substrates based on data from a cell-free proteomics approach.

FEBS Lett 2016 Jan 5;590(2):251-7. Epub 2016 Jan 5.

Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.

Chaperones are essential to maintain the proper folding of various proteins in vivo. The Escherichia coli chaperonin GroEL/GroES (GroE) is one of the best-studied chaperones, and its in vivo substrates have been identified, mainly by mass spectrometry-based proteomic studies. Here, we newly identified 20 in vivo obligate GroE substrates with the aid of data from an in vitro comprehensive analysis. The newly identified substrates have similar physicochemical properties to the known substrates, but their expression levels in vivo were significantly lower. Information from the in vitro comprehensive analysis has the potential to compensate for limitations of the MS-based proteomic approaches.
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http://dx.doi.org/10.1002/1873-3468.12036DOI Listing
January 2016

Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system.

Sci Rep 2015 Dec 15;5:18025. Epub 2015 Dec 15.

Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan.

Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.
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http://dx.doi.org/10.1038/srep18025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678891PMC
December 2015

Large-scale analysis of macromolecular crowding effects on protein aggregation using a reconstituted cell-free translation system.

Front Microbiol 2015 8;6:1113. Epub 2015 Oct 8.

Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology Yokohama, Japan.

Proteins must fold into their native structures in the crowded cellular environment, to perform their functions. Although such macromolecular crowding has been considered to affect the folding properties of proteins, large-scale experimental data have so far been lacking. Here, we individually translated 142 Escherichia coli cytoplasmic proteins using a reconstituted cell-free translation system in the presence of macromolecular crowding reagents (MCRs), Ficoll 70 or dextran 70, and evaluated the aggregation propensities of 142 proteins. The results showed that the MCR effects varied depending on the proteins, although the degree of these effects was modest. Statistical analyses suggested that structural parameters were involved in the effects of the MCRs. Our dataset provides a valuable resource to understand protein folding and aggregation inside cells.
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http://dx.doi.org/10.3389/fmicb.2015.01113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597115PMC
October 2015

Reaction Cycle of Chaperonin GroEL via Symmetric "Football" Intermediate.

Authors:
Hideki Taguchi

J Mol Biol 2015 Sep 18;427(18):2912-8. Epub 2015 Apr 18.

Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Electronic address:

Chaperonin GroEL is an essential chaperone that assists in protein folding in the cell. Since one GroEL ring binds one GroES heptamer, the GroEL double ring permits the formation of two types of GroEL:GroES complexes: asymmetric 1:1 "bullet"-shaped and symmetric 1:2 "football"-shaped GroEL:GroES2 complexes. There have been continuing debates about the mechanism and which complex is critical to the chaperonin-assisted folding. In this review, I summarize the recent progress on the football complex.
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http://dx.doi.org/10.1016/j.jmb.2015.04.007DOI Listing
September 2015
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