Publications by authors named "Hitoshi Nakamoto"

36 Publications

A cyclic lipopeptide surfactin is a species-selective Hsp90 inhibitor that suppresses cyanobacterial growth.

J Biochem 2021 Mar 26. Epub 2021 Mar 26.

Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan.

Heat shock protein 90 (Hsp90) is essential for eukaryotic cells, whereas bacterial homologs play a role under stresses and in pathogenesis. Identifying species-specific Hsp90 inhibitors is challenging because Hsp90 is evolutionarily conserved. We found that a cyclic lipopeptide surfactin inhibits the ATPase activity of Hsp90 from the cyanobacterium Synechococcus elongatus (S. elongatus) PCC 7942 but does not inhibit Escherichia coli (E. coli), yeast and human Hsp90s. Molecular docking simulations indicated that surfactin could bind to the N-terminal dimerization interface of the cyanobacterial Hsp90 in the ATP- and ADP-bound states, which provided molecular insights into the species-selective inhibition. The data suggest that surfactin inhibits a rate-limiting conformational change of S. elongatus Hsp90 in the ATP hydrolysis. Surfactin also inhibited the interaction of the cyanobacterial Hsp90 with a model substrate, and suppressed S. elongatus growth under heat stress, but not that of E. coli. Surfactin did not show significant cellular toxicity toward mammalian cells. These results indicate that surfactin inhibits the cellular function of Hsp90 specifically in the cyanobacterium. The present study shows that a cyclic peptide has a great specificity to interact with a specific homolog of a highly conserved protein family.
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http://dx.doi.org/10.1093/jb/mvab037DOI Listing
March 2021

pH-mediated control of anti-aggregation activities of cyanobacterial and E. coli chaperonin GroELs.

J Biochem 2021 Apr;169(3):351-361

Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.

In contrast to Escherichia coli, cyanobacteria have multiple GroELs, the bacterial homologues of chaperonin/Hsp60. We have shown that cyanobacterial GroELs are mutually distinct and different from E. coli GroEL with which the paradigm for chaperonin structure/function has been established. However, little is known about regulation of cyanobacterial GroELs. This study investigated effect of pH (varied from 7.0 to 8.5) on chaperone activity of GroEL1 and GroEL2 from the cyanobacterium Synechococcus elongatus PCC7942 and E. coli GroEL. GroEL1 and GroEL2 showed pH dependency in suppression of aggregation of heat-denatured malate dehydrogenase, lactate dehydrogenase and citrate synthase. They exhibited higher anti-aggregation activity at more alkaline pHs. Escherichia coli GroEL showed a similar pH-dependence in suppressing aggregation of heat-denatured lactate dehydrogenase. No pH dependence was observed in all the GroELs when urea-denatured lactate dehydrogenase was used for anti-aggregation assay, suggesting that the pH-dependence is related to some denatured structures. There was no significant influence of pH on the chaperone activity of all the GroELs to promote refolding of heat-denatured malate dehydrogenase. It is known that pH in cyanobacterial cytoplasm increases by one pH unit following a shift from darkness to light, suggesting that the pH-change modulates chaperone activity of cyanobacterial GroEL1 and GroEL2.
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http://dx.doi.org/10.1093/jb/mvaa108DOI Listing
April 2021

Regulation of the groESL1 transcription by the HrcA repressor and a novel transcription factor Orf7.5 in the cyanobacterium Synechococcus elongatus PCC7942.

J Gen Appl Microbiol 2020 Jun 10;66(2):85-92. Epub 2020 Apr 10.

Molecular Biology Course, Graduate School of Science and Engineering, Saitama University.

The CIRCE/HrcA system is highly conserved in cyanobacterial genomes. We have shown that heat-shock induction of the groESL1 operon in the cyanobacterium Synechocystis sp. PCC6803 is negatively regulated by the CIRCE/HrcA system. In Synechococcus elongatus PCC7942, a novel heat shock protein, Orf7.5, is involved in positive regulation of the groESL1 transcription. However, Orf7.5 is not conserved in some cyanobacteria, including Synechocystis sp. PCC6803. The purpose of this study is to evaluate the functional conservation of the CIRCE/HrcA system in S. elongatus PCC7942 and to understand the interplay between the CIRCE/HrcA system and the Orf7.5 regulatory system. We constructed single and double mutants of S. elongatus orf7.5, hrcA and orf7.5/hrcA and heat induction of the groESL1 transcription in these mutants was analyzed. Unexpectedly, derepression of the groESL1 transcription in an hrcA mutant was not observed. In all these mutants, the transcription was greatly suppressed under both normal and heat stress conditions, indicating that both HrcA and Orf7.5 are involved in regulation of the groESL1 transcription in a positive way. Consistent with the decrease in the groESL1 mRNA level, all the single and double mutants showed a great loss of acquired thermotolerance. Heat induction of the orf7.5 promoter activity was totally diminished in the orf7.5 mutant, indicating that Orf7.5 activates its own transcription. Yeast two hybrid analysis showed that the principle sigma factor RpoD1 interacts with Orf7.5. These results indicate that Orf7.5 enhances the transcription of groESL1 and orf7.5 by interacting with RpoD1.
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http://dx.doi.org/10.2323/jgam.2020.02.001DOI Listing
June 2020

Evaluation of Open Reduction and Internal Fixation of Mandibular Condyle Fracture by Intraoperative Cone-Beam Computed Tomography in a Hybrid Operating Room.

J Craniofac Surg 2020 May/Jun;31(3):762-765

Division of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital, Takamatsu.

Condylar fractures are the most common fractures of the mandible, and treatment of mandibular condylar fractures by maxillofacial surgeons is a very important procedure. However, the surgical approaches have anatomical limitations. Therefore, it is difficult to evaluate the reduction achieved in open reduction and internal fixation because of the uncertainty in securing a sufficient operative field. As a potential solution, the authors evaluated the benefits of intraoperative cone-beam computed tomography (CBCT) with high image quality performed in a hybrid operating room. Intraoperative CBCT is easy to perform in a hybrid operating room, and it is possible to quickly evaluate high-quality CT images, including 3D images. Because the state of reduction of mandibular condylar fractures also affects the prognosis of treatment, more precise reduction and fixation should improve prognoses. The use of CBCT in a hybrid operating room also avoids re-operation, and patients benefit from minimum invasive surgery. Intraoperative CBCT is a very useful strategy for evaluation of mandibular condylar fracture surgical treatment.
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http://dx.doi.org/10.1097/SCS.0000000000006101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329199PMC
September 2020

Stimulation of the ATPase activity of Hsp90 by zerumbone modification of its cysteine residues destabilizes its clients and causes cytotoxicity.

Biochem J 2018 08 16;475(15):2559-2576. Epub 2018 Aug 16.

Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

Hsp90 is an ATP-dependent molecular chaperone that assists folding and conformational maturation/maintenance of many proteins. It is a potential cancer drug target because it chaperones oncoproteins. A prokaryotic homolog of Hsp90 (HtpG) is essential for thermo-tolerance in some bacteria and virulence of zoonotic pathogens. To identify a new class of small molecules which target prokaryotic and eukaryotic Hsp90s, we studied the effects of a naturally occurring cyclic sesquiterpene, zerumbone, which inhibits proliferation of a wide variety of tumor cells, on the activity of Hsp90. Zerumbone enhanced the ATPase activity of cyanobacterial Hsp90 (Hsp90), yeast Hsp90, and human Hsp90α. It also enhanced the catalytic efficiency of Hsp90 by greatly increasing Mass analysis showed that zerumbone binds to cysteine side chains of Hsp90 covalently. Mutational studies identified 3 cysteine residues (one per each domain of Hsp90) that are involved in the enhancement, suggesting the presence of allosteric sites in the middle and C-terminal domains of Hsp90 Treatment of cyanobacterial cells with zerumbone caused them to become very temperature-sensitive, a phenotype reminiscent of cyanobacterial Hsp90 mutants, and also decreased the cellular level of linker polypeptides that are clients for Hsp90 Zerumbone showed cellular toxicity on cancer-derived mammalian cells by inducing apoptosis. In addition, zerumbone inhibited the binding of Hsp90/Cdc37 to client kinases. Altogether, we conclude that modification of cysteine residues of Hsp90 by zerumbone enhances its ATPase activity and inhibits physiological Hsp90 function. The activation of Hsp90 may provide new strategies to inhibit its chaperone function in cells.
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http://dx.doi.org/10.1042/BCJ20180230DOI Listing
August 2018

Non-housekeeping, non-essential GroEL (chaperonin) has acquired novel structure and function beneficial under stress in cyanobacteria.

Physiol Plant 2017 Nov 20;161(3):296-310. Epub 2017 Jul 20.

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.

GroELs which are prokaryotic members of the chaperonin (Cpn)/Hsp60 family are molecular chaperones of which Escherichia coli GroEL is a model for subsequent research. The majority of bacterial species including E. coli and Bacillus subtilis have only one essential groEL gene that forms an operon with the co-chaperone groES gene. In contrast to these model bacteria, two or three groEL genes exist in cyanobacterial genomes. One of them, groEL2, does not form an operon with the groES gene, whereas the other(s) does. In the case of cyanobacteria containing two GroEL homologs, one of the GroELs, GroEL1, substitutes for the native GroEL in an E. coli cell, but GroEL2 does not. Unlike the E. coli GroEL, GroEL2 is not essential, but it plays an important role which is not substitutable by GroEL1 under stress. Regulation of expression and biochemical properties of GroEL2 are different/diversified from GroEL1 and E. coli GroEL in many aspects. We postulate that the groEL2 gene has acquired a novel, beneficial function especially under stresses and become preserved by natural selection, with the groEL1 gene retaining the original, house-keeping function. In this review, we will focus on difference between the two GroELs in cyanobacteria, and divergence of GroEL2 from the E. coli GroEL. We will also compare cyanobacterial GroELs with the chloroplast Cpns (60α and 60β) which are thought to be evolved from the cyanobacterial GroEL1. Chloroplast Cpns appear to follow the different path from cyanobacterial GroELs in the evolution after gene duplication of the corresponding ancestral groEL gene.
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http://dx.doi.org/10.1111/ppl.12595DOI Listing
November 2017

Goniothalamin enhances the ATPase activity of the molecular chaperone Hsp90 but inhibits its chaperone activity.

J Biochem 2015 Mar 7;157(3):161-8. Epub 2014 Oct 7.

Molecular Biology Course, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan; Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia; and Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

Hsp90 is an ATP-dependent molecular chaperone that is involved in important cellular pathways such as signal transduction pathways. It is a potential cancer drug target because it plays a critical role for stabilization and activation of oncoproteins. Thus, small molecule compounds that control the Hsp90 function are useful to elucidate potential lead compounds against cancer. We studied effect of a naturally occurring styryl-lactone goniothalamin on the activity of Hsp90. Although many drugs targeting Hsp90 inhibit the ATPase activity of Hsp90, goniothalamin enhanced rather than inhibited the ATPase activity of a cyanobacterial Hsp90 (HtpG) and a yeast Hsp90. It increased both K(m) and k(cat) of the Hsp90s. Domain competition assays and tryptophan fluorescence measurements with various truncated derivatives of HtpG indicated that goniothalamin binds to the N-terminal domain of HtpG. Goniothalamin did not influence on the interaction of HtpG with a non-native protein or the anti-aggregation activity of HtpG significantly. However, it inhibited the activity of HtpG that assists refolding of a non-native protein in cooperation with the Hsp70 chaperone system. This is the first report to show that a small molecule that binds to the N-terminal domain of Hsp90 activates its ATPase activity, while inhibiting the chaperone function of Hsp90.
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http://dx.doi.org/10.1093/jb/mvu061DOI Listing
March 2015

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

J Biol Chem 2014 Feb 12;289(9):6110-9. Epub 2014 Jan 12.

From the Department of Biochemistry and Molecular Biology and.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.
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http://dx.doi.org/10.1074/jbc.M113.524801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937677PMC
February 2014

Molecular chaperones as drug targets.

Curr Pharm Des 2013 ;19(3):307-8

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http://dx.doi.org/10.2174/138161213804143671DOI Listing
October 2013

The therapeutic target Hsp90 and cancer hallmarks.

Curr Pharm Des 2013 ;19(3):347-65

Department of Cell & Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.

Hsp90 is a major molecular chaperone that is expressed abundantly and plays a pivotal role in assisting correct folding and functionality of its client proteins in cells. The Hsp90 client proteins include a wide variety of signal transducing molecules such as protein kinases and steroid hormone receptors. Cancer is a complex disease, but most types of human cancer share common hallmarks, including self-sufficiency in growth signals, insensitivity to growth-inhibitory mechanism, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. A surprisingly large number of Hsp90-client proteins play crucial roles in establishing cancer cell hallmarks. We start the review by describing the structure and function of Hsp90 since conformational changes during the ATPase cycle of Hsp90 are closely related to its function. Many co-chaperones, including Hop, p23, Cdc37, Aha1, and PP5, work together with Hsp90 by modulating the chaperone machinery. Post-translational modifications of Hsp90 and its cochaperones are vital for their function. Many tumor-related Hsp90-client proteins, including signaling kinases, steroid hormone receptors, p53, and telomerase, are described. Hsp90 and its co-chaperones are required for the function of these tumor-promoting client proteins; therefore, inhibition of Hsp90 by specific inhibitors such as geldanamycin and its derivatives attenuates the tumor progression. Hsp90 inhibitors can be potential and effective cancer chemotherapeutic drugs with a unique profile and have been examined in clinical trials. We describe possible mechanisms why Hsp90 inhibitors show selectivity to cancer cells even though Hsp90 is essential also for normal cells. Finally, we discuss the "Hsp90-addiction" of cancer cells, and suggest a role for Hsp90 in tumor evolution.
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http://dx.doi.org/10.2174/138161213804143725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553218PMC
October 2013

Heat shock response in photosynthetic organisms: membrane and lipid connections.

Prog Lipid Res 2012 Jul 29;51(3):208-20. Epub 2012 Mar 29.

Institute of Biochemistry, Biol. Res. Centre, Hungarian Acad. Sci., Temesvári krt. 62, H-6734 Szeged, Hungary.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.
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http://dx.doi.org/10.1016/j.plipres.2012.02.002DOI Listing
July 2012

Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity.

Biochem J 2011 Apr;435(1):237-46

Molecular Biology Course, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.
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http://dx.doi.org/10.1042/BJ20100743DOI Listing
April 2011

Comparative biochemical characterization of two GroEL homologs from the cyanobacterium Synechococcus elongatus PCC 7942.

Biosci Biotechnol Biochem 2010 7;74(11):2273-80. Epub 2010 Nov 7.

Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Japan.

Unlike Escherichia coli, cyanobacteria generally contain two GroEL homologs. The chaperone function of cyanobacterial GroELs was examined in vitro for the first time with GroEL1 and GroEL2 of Synechococcus elongatus PCC 7942. Both GroELs prevented aggregation of heat-denatured proteins. The ATPase activity of GroEL1 was approximately one-sixth that of Escherichia coli GroEL, while that of GroEL2 was insignificant. The activities of both GroELs were enhanced by GroES, while that of Escherichia coli GroEL was suppressed. The ATPase activity of GroEL1 was greatly enhanced in the presence of GroEL2, but the folding activities of GroEL1 and GroEL2 were much lower than that of Escherichia coli GroEL, regardless of the co-presence of the counterpart or GroES. Both native and recombinant GroEL1 forms a tetradecamer like Escherichia coli GroEL, while GroEL2 forms a heptamer or dimer, but the GroEL1 and GroEL2 oligomers were extremely unstable. In sum, we concluded that the cyanobacterial GroELs are mutually distinct and different from Escherichia coli GroEL.
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http://dx.doi.org/10.1271/bbb.100493DOI Listing
March 2011

HtpG, the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942.

Mol Microbiol 2010 May 25;76(3):576-89. Epub 2010 Mar 25.

Department of Biochemistry and Molecular Biology, Saitama University, Saitama 338-8570, Japan.

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain-truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N-terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C-terminal domain did not. Truncation of the C-terminal domain that is involved in the dimerization of HtpG led to decrease in the anti-aggregation activity, while fusion of the N-terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti-aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07139.xDOI Listing
May 2010

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

Biochem Biophys Res Commun 2009 Mar 23;380(3):520-4. Epub 2009 Jan 23.

Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H2O2. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.
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http://dx.doi.org/10.1016/j.bbrc.2009.01.091DOI Listing
March 2009

A small heat-shock protein confers stress tolerance and stabilizes thylakoid membrane proteins in cyanobacteria under oxidative stress.

Arch Microbiol 2009 Apr 24;191(4):319-28. Epub 2009 Jan 24.

Department of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan.

Small heat-shock proteins are molecular chaperones that bind and prevent aggregation of nonnative proteins. They also associate with membranes. In this study, we show that the small heat-shock protein HspA plays a protective role under oxidative stress in the cyanobacterium Synechococcus elongatus strain ECT16-1, which constitutively expresses HspA. Compared with the reference strain ECT, ECT16-1 showed much better growth and viability in the presence of hydrogen peroxide. Under the peroxide stress, pigments in thylakoid membrane, chlorophyll, carotenoids, and phycocyanins, were continuously reduced in ECT, but in ECT16-1 they decreased only during the first 24 h of stress; thereafter no further reduction was observed. For comparison, we analyzed a wild type and an hspA deletion strain from Synechocystis sp. PCC 6803 and found that lack of hspA significantly affected the viability of the cell and the pigment content in the presence of methyl viologen, suggesting that HspA stabilizes membrane proteins such as the photosystems and phycobilisomes from oxidative damage. In vitro pull down assays showed a direct interaction of HspA with components of phycobilisomes. These results show that HspA and small heat-shock proteins in general play an important role in the acclimation to oxidative stress in cyanobacteria.
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http://dx.doi.org/10.1007/s00203-009-0457-zDOI Listing
April 2009

Expression and function of a groEL paralog in the thermophilic cyanobacterium Thermosynechococcus elongatus under heat and cold stress.

FEBS Lett 2008 Oct 9;582(23-24):3389-95. Epub 2008 Sep 9.

Department of Biochemistry and Molecular Biology, Saitama University, Saitama 338-8570, Japan.

Cyanobacterial genomes generally contain two groEL genes, referred to as groEL1 and groEL2. The purpose of this study is to elucidate a role of groEL2 in the adaptation of the thermophilic cyanobacterium Thermosynechococcuselongatus to hot environments. Both groEL genes were found to be heat-induced, while only groEL2 was greatly cold-induced. Primer extension and gel mobility shift analyses indicated that transcriptional regulation of groEL2 is different from that of groESL1. The groEL2 gene was dispensable under normal growth conditions at 50 degrees C as a groEL2 disruptant was viable. This groEL2 mutant was highly sensitive to both high and low temperatures.
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http://dx.doi.org/10.1016/j.febslet.2008.08.034DOI Listing
October 2008

Interaction of the molecular chaperone HtpG with uroporphyrinogen decarboxylase in the cyanobacterium Synechococcus elongatus PCC 7942.

Biosci Biotechnol Biochem 2008 May 7;72(5):1394-7. Epub 2008 May 7.

Department of Biochemistry and Molecular Biology, Saitama University, Japan.

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.
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http://dx.doi.org/10.1271/bbb.80093DOI Listing
May 2008

Membrane regulation of the stress response from prokaryotic models to mammalian cells.

Ann N Y Acad Sci 2007 Oct 26;1113:40-51. Epub 2007 Jul 26.

Institute of Biochemistry, Biology Research Centre, Hungarian Academy of Sciences,Temesvari krt 62, H-6726, Szeged, Hungary.

"Membrane regulation" of stress responses in various systems is widely studied. In poikilotherms, membrane rigidification could be the first reaction to cold perception: reducing membrane fluidity of membranes at physiological temperatures is coupled with enhanced cold inducibility of a number of genes, including desaturases (see J.L. Harwood's article in this Proceedings volume). A similar role of changes in membrane physical state in heat (oxidative stress, etc.) sensing- and signaling gained support recently from prokaryotes to mammalian cells. Stress-induced remodeling of membrane lipids could influence generation, transduction, and deactivation of stress signals, either through global effects on the fluidity of the membrane matrix, or by specific interactions of boundary (or raft) lipids with receptor proteins, lipases, ion channels, etc. Our data point to membranes not only as targets of stress, but also as sensors in activating a stress response.
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http://dx.doi.org/10.1196/annals.1391.027DOI Listing
October 2007

A novel light- and heat-responsive regulation of the groE transcription in the absence of HrcA or CIRCE in cyanobacteria.

FEBS Lett 2007 May 9;581(9):1871-80. Epub 2007 Apr 9.

Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

The inactivation of the hrcA gene resulted in de-repression of the two CIRCE-containing groE genes in a cyanobacterium Synechocystis sp. strain PCC6803, indicating that the CIRCE operator/HrcA repressor system operates in the cyanobacterium. We found that the groE expression in the hrcA mutant is greatly induced by heat and/or light. Removal of a K-box containing and an N-box containing region upstream of the groESL1 promoter abolished light-induced transcription of a luxAB reporter gene fused with the groESL1 promoter. Similar sequences to the K-box, GTTCGG-NNAN-CCNNAC, were also found upstream of the dnaK2 genes. A specific binding of a protein(s) to the N-box, GATCTA, was detected by a gel mobility shift assay with using cell extracts. We propose that the cyanobacterial groEL expression is regulated by a putative positive mechanism mediated by these novel elements in addition to the HrcA/CIRCE system. The groEL2 genes from Synechococcus sp. strain PCC 7942 and Thermosynechococcus elongatus, which lack CIRCE, K-box, and N-box naturally, were also induced by heat and/or light, indicating that the control mechanism of the unique light-responsive groE expression is highly diversified in cyanobacteria.
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http://dx.doi.org/10.1016/j.febslet.2007.03.084DOI Listing
May 2007

Studies on the role of HtpG in the tetrapyrrole biosynthesis pathway of the cyanobacterium Synechococcus elongatus PCC 7942.

Biochem Biophys Res Commun 2007 Jan 3;352(1):36-41. Epub 2006 Nov 3.

Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan.

In cyanobacterium Synechococcus elongatus PCC 7942, we observed that htpG-overexpression caused remarkable growth inhibition. In addition, subcellular fractionation experiments showed that HtpG was localized in the membrane fraction. To understand its function in cyanobacteria, we carried out yeast two-hybrid screening to identify specific proteins interacting with HtpG, and found out, HemE, uroporphyrinogen decarboxylase. When compared to the wild-type strain, the htpG-null mutant and -overexpressing strains exhibited higher and lower cytosolic HemE activity, based on the coproporphyrin production, respectively. These results strongly suggest that HtpG is involved in the regulation of tetrapyrrole biosynthesis through interacting with HemE protein.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.144DOI Listing
January 2007

Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase.

Photochem Photobiol 2006 Nov-Dec;82(6):1677-85

Bioengineering, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.
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http://dx.doi.org/10.1562/2006-05-07-RA-893DOI Listing
April 2007

Interaction of a small heat shock protein with light-harvesting cyanobacterial phycocyanins under stress conditions.

FEBS Lett 2006 May 27;580(13):3029-34. Epub 2006 Apr 27.

Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-Okubo, Saitama 338-8570, Japan.

Phycobiliproteins such as phycocyanins are the most abundant proteins found in cyanobacteria which are assembled to form the phycobilisome. Here, we showed that a small heat shock protein, HspA, interacts directly with phycocyanins from the cyanobacterium Synechococcus sp. strain PCC 7942 in vitro and suppresses inactivation of their light-harvesting functions due to heat denaturation in the presence of hydrogen peroxide. Under the denaturing conditions, phycobilisomes were de-assembled to lighter complexes and then aggregated. HspA associated with phycocyanins in the dissociated complexes, and suppressed the aggregation. The specific interaction between a small heat shock protein and phycocyanins was further supported by the fact that HspA and alpha-crystallin protected isolated phycocyanins from denaturation, while HtpG and lysozyme did not. The maximum protection was observed at a molar ratio of four HspA monomer per one phycocyanin (alpha beta) monomer.
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http://dx.doi.org/10.1016/j.febslet.2006.04.047DOI Listing
May 2006

Light-driven hydrogen production by a hybrid complex of a [NiFe]-hydrogenase and the cyanobacterial photosystem I.

Photochem Photobiol 2006 May-Jun;82(3):676-82

Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama, Japan.

In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.
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http://dx.doi.org/10.1562/2006-01-16-RA-778DOI Listing
July 2006

Intact carboxysomes in a cyanobacterial cell visualized by hilbert differential contrast transmission electron microscopy.

J Bacteriol 2006 Jan;188(2):805-8

Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.
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http://dx.doi.org/10.1128/JB.188.2.805-808.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1347272PMC
January 2006

Constitutive expression of small heat shock protein in an htpG disruptant of the Cyanobacterium Synechococcus sp. PCC 7942.

Curr Microbiol 2005 May 11;50(5):272-6. Epub 2005 Apr 11.

Department of Biochemistry and Molecular Biology, Saitama University, Saitama, 338-8570, Japan.

In cyanobacteria, a disruptant of hspA encoding a small heat shock protein homologue, shows decreased cell growth rates at moderately high temperatures, and loss of both basal and acquired thermo-tolerances, which resemble the phenotype of an htpG disruptant. In vitro studies have shown that both small heat shock protein and Hsp90 can bind and keep non-native proteins in a refolding-competent state under denaturing conditions. The aim of the present study is to elucidate whether constitutive expression of HspA can functionally replace HtpG, a prokaryotic homolog of Hsp90, in the cyanobacterium Synechococcus sp. PCC 7942. HspA did not improve the viability of the htpG disruptant at a lethal temperature, although it did that of the wild type. It did not improve an iron-starved phenotype of the mutant under normal growth conditions, a novel phenotype found in the present study. These results suggest that cellular function of HtpG may differ significantly from that of HspA.
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http://dx.doi.org/10.1007/s00284-005-4486-9DOI Listing
May 2005

Post-transcriptional control of the cyanobacterial hspA heat-shock induction.

Biochem Biophys Res Commun 2005 Jun;331(2):583-8

Department of Biochemistry and Molecular Biology, Saitama University, Saitama 338-8570, Japan.

The hspA gene encodes a small heat-shock protein in the thermophilic cyanobacterium Synechococcus vulcanus. To gain insight into the post-transcriptional regulation, hspA was expressed in Escherichia coli. HspA was induced upon a temperature upshift from 30 to 42 degrees C, although the hspA transcription in E. coli occurred constitutively at both 30 and 42 degrees C. Neither replacement of the native hspA promoter with the lacZ promoter nor the addition of rifampin abolished the heat induction. Thus, the primary form of regulation of the heat induction is at the post-transcriptional level. Analyses of expression of a series of the transcriptional and translational hspA-lacZ fusions confirmed the constitutive transcription, and demonstrated that the heat induction occurred only in the translational fusions. They further indicated the presence of regulatory elements involved in the translational regulation. An element in the 5'-untranslated region of the hspA mRNA suppressed the translation, while that in the hspA coding region was required for the de-repression of the translation and for thermal regulation.
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http://dx.doi.org/10.1016/j.bbrc.2005.04.009DOI Listing
June 2005

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria.

FEBS Lett 2005 Feb 26;579(5):1235-42. Epub 2005 Jan 26.

Department of Regulation-Biology, Saitama University, Saitama 338-8570, Japan.

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.
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http://dx.doi.org/10.1016/j.febslet.2004.12.095DOI Listing
February 2005

Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm.

Biochim Biophys Acta 2004 Nov;1694(1-3):111-9

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830, North University Dr., Ann Arbor, MI 48109-1048, USA.

Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source.
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http://dx.doi.org/10.1016/j.bbamcr.2004.02.012DOI Listing
November 2004

Comparative analysis of the hspA mutant and wild-type Synechocystis sp. strain PCC 6803 under salt stress: evaluation of the role of hspA in salt-stress management.

Arch Microbiol 2004 Dec 12;182(6):487-97. Epub 2004 Oct 12.

Department of Biochemistry and Molecular Biology, Saitama University, Saitama 338-8570, Japan.

DNA microarray analysis has previously revealed that hspA, which encodes a small heat-shock protein, is the second most highly expressed gene under salt stress in Synechocystis sp. strain PCC 6803. Consequently, an hspA deletion mutant was studied under various salt stresses in order to identify a potential role of HspA in salt stress management. The mutant had a growth disadvantage under moderate salt stress. It lost the ability to develop tolerance to a lethal salt treatment by a moderate salt pre-treatment when the tolerance was evaluated by cell survival and the level of major soluble proteins, phycocyanins, while the wild-type acquired tolerance. Under various salt stresses, the mutant failed to undergo the ultrastructural changes characteristic of wild-type cells. The mutant, which showed higher survival than the wild-type after a direct shift to lethal salt conditions, accumulated higher levels of groESL1 and groEL2 transcripts and the corresponding proteins, GroES, GroEL1, and GroEL2, suggesting a role for these heat-shock proteins in conferring basal salt tolerance. Under salt stress, heat-shock genes, such as hspA, groEL2, and dnaK2, were transcriptionally induced and greatly stabilized, indicating a transcriptional and post-transcriptional mechanism of acclimation to salt stress involving these heat-shock genes.
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http://dx.doi.org/10.1007/s00203-004-0733-xDOI Listing
December 2004