記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Molecular Sciences  

Zinc transporters take up/release zinc ions (Zn2+) across biological membranes and maintain intracellular and intra-organellar Zn2+ homeostasis. Since this process requires a series of conformational changes in the transporters, detailed information about the structures of different reaction intermediates is required for a comprehensive understanding of their Zn2+ transport mechanisms. Recently, various Zn2+ transport systems have been identified in bacteria, yeasts, plants, and humans. Based on structural analyses of human ZnT7, human ZnT8, and bacterial YiiP, we propose updated models explaining their mechanisms of action to ensure efficient Zn2+ transport. We place particular focus on the mechanistic roles of the histidine-rich loop shared by several zinc transporters, which facilitates Zn2+ recruitment to the transmembrane Zn2+-binding site. This review provides an extensive overview of the structures, mechanisms, and physiological functions of zinc transporters in different biological kingdoms.

DOI： 10.3390/ijms25053045

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その他リンク： https://www.mdpi.com/1422-0067/25/5/3045

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Communications Chemistry  

Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S’s) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 μg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.

DOI： 10.1038/s42004-023-01056-4

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その他リンク： https://www.nature.com/articles/s42004-023-01056-4

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.

DOI： 10.1016/j.jbc.2023.105274

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+-bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a dimer via tight interactions in both the cytosolic and transmembrane (TM) domains of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to the TM metal transport pathway. These structures permit mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.

DOI： 10.1038/s41467-023-40521-5

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

DOI： 10.1038/s41467-023-40182-4

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その他リンク： https://www.nature.com/articles/s41467-023-40182-4

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Reports  

The endoplasmic reticulum (ER) maintains an oxidative redox environment that is advantageous for the oxidative folding of nascent polypeptides entering the ER. Reductive reactions within the ER are also crucial for maintaining ER homeostasis. However, the mechanism by which electrons are supplied for the reductase activity within the ER remains unknown. Here, we identify ER oxidoreductin-1α (Ero1α) as an electron donor for ERdj5, an ER-resident disulfide reductase. During oxidative folding, Ero1α catalyzes disulfide formation in nascent polypeptides through protein disulfide isomerase (PDI) and then transfers the electrons to molecular oxygen via flavin adenine dinucleotide (FAD), ultimately yielding hydrogen peroxide (H2O2). Besides this canonical electron pathway, we reveal that ERdj5 accepts electrons from specific cysteine pairs in Ero1α, demonstrating that the oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER. Moreover, this electron transfer pathway also contributes to maintaining ER homeostasis by reducing H2O2 production in the ER.

DOI： 10.1016/j.celrep.2023.112742

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記述言語：英語   出版者・発行元：Nature Communications  

The original version of this article contained a duplication of Supplementary Fig. 1 in the Supplementary Information. The Supplementary Information file has been corrected.

DOI： 10.1038/s41467-023-39273-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

DOI： 10.1038/s41467-023-38397-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Science Advances  

Secretory pathway Ca2+/Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo–electron microscopy structures of human SPCA1a in the ATP and Ca2+/Mn2+-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion–binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+-binding site in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+/Mn2+ transport.

DOI： 10.1126/sciadv.add9742

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Reports  

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca2+-bound phosphorylated (E1P·2Ca2+) and Ca2+-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca2+ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca2+ and that the Ca2+ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.

DOI： 10.1016/j.celrep.2022.111760

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：日本語  

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BioEssays  

Sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), a member of the SERCA family, is expressed ubiquitously and transports Ca2+ into the sarco/endoplasmic reticulum using the energy provided by ATP binding and hydrolysis. The crystal structure of SERCA2b in its Ca2+- and ATP-bound (E1∙2Ca2+-ATP) state and cryo-electron microscopy (cryo-EM) structures of the protein in its E1∙2Ca2+-ATP and Ca2+-unbound phosphorylated (E2P) states have provided essential insights into how the overall conformation and ATPase activity of SERCA2b is regulated by the transmembrane helix 11 and the subsequent luminal extension loop, both of which are specific to this isoform. More recently, our cryo-EM analysis has revealed that SERCA2b likely adopts open and closed conformations of the cytosolic domains in the Ca2+-bound but ATP-free (E1∙2Ca2+) state, and that the closed conformation represents a state immediately prior to ATP binding. This review article summarizes the unique mechanisms underlying the conformational and functional regulation of SERCA2b.

DOI： 10.1002/bies.202200052

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Structural basis of the conformational and functional regulation of human SERCA2b, the ubiquitous endoplasmic reticulum calcium pump

記述言語：英語  

Active Expression of Genes for Protein Modification Enzymes in Habu Venom Glands

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACS Sensors  

Although many Zn2+fluorescent probes have been developed, there remains a lack of consensus on the labile Zn2+concentrations ([Zn2+]) in several cellular compartments, as the fluorescence properties and zinc affinity of the fluorescent probes are greatly affected by the pH and redox environments specific to organelles. In this study, we developed two turn-on-type Zn2+fluorescent probes, namely, ZnDA-2H and ZnDA-3H, with low pH sensitivity and suitable affinity (Kd= 5.0 and 0.16 nM) for detecting physiological labile Zn2+in various cellular compartments, such as the cytosol, nucleus, ER, and mitochondria. Due to their sufficient membrane permeability, both probes were precisely localized to the target organelles in HeLa cells using HaloTag labeling technology. Using an in situ standard quantification method, we identified the [Zn2+] in the tested organelles, resulting in the subcellular [Zn2+] distribution as [Zn2+]ER< [Zn2+]mito< [Zn2+]cyto∼[Zn2+]nuc.

DOI： 10.1021/acssensors.1c02153

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization.
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 Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.

DOI： 10.1016/j.str.2021.03.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cryo‐EM analysis provides new mechanistic insight into ATP binding to Ca2+‐ATPase SERCA2b
Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+ . We propose a novel mechanism of ATP binding to SERCA2b.

DOI： 10.15252/embj.2021108482

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding
P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology.

DOI： 10.3390/biology10111112

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.

DOI： 10.3390/molecules26102853

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.isci.2021.102296

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Time-resolved single-molecule observations by high-speed atomic force microscopy (HS-AFM), have greatly advanced our understanding of how proteins operate to fulfill their unique functions. Using this device, we succeeded in visualizing two members of the protein disulfide isomerase family (PDIs) that act to catalyze oxidative folding and reductive unfolding in the endoplasmic reticulum (ER). ERdj5, an ER-resident disulfide reductase that promotes ER-associated degradation, reduces nonnative disulfide bonds of misfolded proteins utilizing the dynamics of its N-terminal and C-terminal clusters. With unfolded substrates, canonical PDI assembles to form a face-to-face dimer with a central hydrophobic cavity and multiple redox-active sites to accelerate oxidative folding inside the cavity. Altogether, PDIs exert highly dynamic mechanisms to ensure the protein quality control in the ER.

DOI： 10.1016/j.sbi.2020.10.004

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PDI family members as guides for client folding and assembly

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine's impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced β-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.

DOI： 10.7554/eLife.60970

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Fluorescent Zn2+ probes used for the quantitative analysis of labile Zn2+ concentration ([Zn2+]) in target organelles are crucial for understanding the role of Zn2+ in biological processes. Although several fluorescent Zn2+ probes have been developed to date, there is still a lack of consensus concerning the [Zn2+] in intracellular organelles. In this study, we describe the development of ZnDA-1H, a small-molecule fluorescent probe for Zn2+, which exhibits less pH sensitivity, high Zn2+ selectivity, and large fluorescence enhancement upon binding to Zn2+. Through protein labeling technology, ZnDA-1H was precisely targeted in various intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. ZnDA-1H exhibited a reversible fluorescence response toward labile Zn2+ in these organelles in live cells. Using this probe, the [Zn2+] in the Golgi apparatus was estimated to be 25 ± 1 nM, suggesting that labile Zn2+ plays a physiological role in the secretory pathway.

DOI： 10.1016/j.chembiol.2020.09.003

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+-adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3- states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

DOI： 10.1126/sciadv.abb0147

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Oxidative protein folding occurs primarily in the mammalian endoplasmic reticulum (ER), enabled by a diverse network comprising more than 20 members of the protein disulfide isomerase (PDI) family and more than five PDI oxidases. While the canonical disulfide bond formation pathway involving Ero1α and PDI has been well studied so far, the physiological roles of the newly identified PDI oxidases, glutathione peroxidase-7 (GPx7) and -8 (GPx8), are only poorly understood. We here demonstrated that human GPx7 has much higher reactivity with H₂O₂ and hence greater PDI oxidation activity than human GPx8. The high reactivity of GPx7 is due to the presence of a catalytic tetrad at the redox active site, which stabilizes the sulfenylated species generated upon the reaction with H₂O₂. While it was previously postulated that GPx7 catalysis involved a highly reactive peroxidatic cysteine that can be sulfenylated by H₂O₂, we revealed that a resolving cysteine instead regulates the PDI oxidation activity of GPx7. We also determined that GPx7 formed complexes preferentially with PDI and P5 in H₂O₂-treated cells. Altogether, these results suggest that human GPx7 functions as an H₂O₂-dependent PDI oxidase in cells whereas PDI oxidation may not be the central physiological role of human GPx8.

DOI： 10.1074/jbc.ra120.013607

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

DOI： 10.1073/pnas.2004606117

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The protein disulfide isomerase family: from proteostasis to pathogenesis

記述言語：英語   掲載種別：研究論文（学術雑誌）  

High-resolution crystal structure of Arabidopsis FLOWERING LOCUS T illuminates its phospholipid-binding site in flowering

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Structural basis of sarco/endoplasmic reticulum Ca2+-ATPase 2b regulation via transmembrane helix interplay

DOI： 10.1016/j.celrep.2019.03.106

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Zinc regulates ERp44-dependent protein quality control in the early secretory pathway

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Coupling effects of thiol and urea-type groups for promotion of oxidative protein folding
Coupling of thiol and urea-type-NHC(═X)NH2 (X = O or NH) groups is effective in promoting oxidative protein folding. In particular, a thiol compound coupled with a guanidyl (X = NH) group significantly accelerates the rates of folding processes and enhances the yields of native proteins.

DOI： 10.1039/c8cc08657e

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Methods to identify the substrates of thiol-disulfide oxidoreductases

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Identification of the physiological substrates of PDIp, a pancreas-specific protein disulfide isomerase family member protein, from a mouse tissue
About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo, revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.

DOI： 10.1074/jbc.RA118.003694

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ero1-mediated reoxidation of PDI accelerates the folding of cone snail

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Characterization and optimization of two-chain folding pathways of insulin via native chain assembly
Until recently the total synthesis of insulin, with its characteristic heterodimeric structure crosslinked by two interchain and one intrachain disulfide (SS) bridge, remained largely an unsolved challenge. By optimizing the synthesis and directed disulfide crosslinking of the two chains, and by applying biomimetic monocomponent proinsulin approaches, efficient insulin syntheses have been realized. Here we report the optimization and characterisation of an alternative strategy, oxidative native chain assembly. In this method unprotected A- and B-chains assemble oxidatively under thermodynamic control to afford bovine pancreatic insulin in 39% yield. Folding is found to proceed predominantly via structured 1SS(star) and 2SS(star) intermediates with a common interchain Cys(A20)-Cys(B19) disulfide. These results suggest that native chain assembly, long considered inefficient, may represent a reasonable strategy to access insulin variants. This is supported by the synthesis of human insulin and human type-II relaxin in yields of up to 49 and 47%, respectively, although the application to human insulin Val(A16) variant is unsuccessful.

DOI： 10.1038/s42004-018-0024-0

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

DOI： 10.1038/s41467-017-01311-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.

DOI： 10.1016/j.str.2017.04.001

記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ange.201701654

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U(A), C7U(B)] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A-and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (t(1/2) approximate to 8 h vs. approximate to 1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.

DOI： 10.1002/anie.201701654

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERp44 retrieves some endoplasmic reticulum (ER)-resident enzymes and immature oligomers of secretory proteins from the Golgi. Association of ERp44 with its clients is regulated by pH-dependent mechanisms, but the molecular details are not fully understood. Here we report high-resolution crystal structures of human ERp44 at neutral and weakly acidic pH. These structures reveal key regions in the C-terminal tail (C tail) missing in the original crystal structure, including a regulatory histidine-rich region and a subsequent extended loop. The former region forms a short alpha-helix (alpha 16), generating a histidine-clustered site (His cluster). At low pH, the three Trx-like domains of ERp44 ("a," "b," and "b'") undergo significant rearrangements, likely induced by protonation of His157 located at the interface between the a and b domains. The alpha 16-helix is partially unwound and the extended loop is disordered in weakly acidic conditions, probably due to electrostatic repulsion between the protonated histidines in the His cluster. Molecular dynamics simulations indicated that helix unwinding enhances the flexibility of the C tail, disrupting its normal hydrogen-bonding pattern. The observed pH-dependent conformational changes significantly enlarge the positively charged regions around the client-binding site of ERp44 at low pH. Mutational analyses showed that ERp44 forms mixed disulfides with specific cysteines residing on negatively charged loop regions of Ero1 alpha. We propose that the protonation states of the essential histidines regulate the ERp44-client interaction by altering the C-tail dynamics and surface electrostatic potential of ERp44.

DOI： 10.1073/pnas.1621426114

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In the mammalian endoplasmic reticulum, oxidoreductin-1 (Ero1) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O-2 to H2O2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H2O2 generation cause cell death, Ero1 activity must be properly regulated. In addition to the four catalytic cysteines (Cys(94), Cys(99), Cys(104), and Cys(131)) that are located in the flexible active site region, the Cys(208)-Cys(241) pair located at the base of another flexible loop is necessary for Ero1 regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys(208)-Cys(241) disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys(208) and Cys(241) did not grossly affect the thermal stability or overall shape of Ero1, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys(208) and Cys(241) provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1 through reduction of the Cys(208)-Cys(241) disulfide. We propose a mechanism of dual Ero1 regulation by dynamic redox interactions between PDI and the two Ero1 flexible loops that harbor the regulatory cysteines.

DOI： 10.1074/jbc.M116.735662

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+](i)) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+](ER)), whereas a higher [Ca2+](ER) induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+](ER)-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

DOI： 10.1073/pnas.1605818113

記述言語：英語   掲載種別：研究論文（学術雑誌）  

8638

DOI： 10.1016/j.redox.2015.11.004

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcus sp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.

DOI： 10.1042/BJ20151223

記述言語：英語   掲載種別：研究論文（学術雑誌）  

One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg2+ and Ca2+ after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg2+ or Ca2+, indicating that the divalent cations accelerate the sliding likely by weakening the DNA p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg2+ and Ca2+, which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2015.06.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O-2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1 alpha (Ero1 alpha-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the Flavin cofactor where O-2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/cy-(241) - dependent mixed-disulfide complex with Ero1 alpha. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O-2 diffusion simulations, these data describe the first enzymatically controlled O-2 access into a flavoprotein active site, provide molecular-level understanding of En:Act regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2015.02.011

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein-protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2015.02.010

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB(AT)) with Ni ions. Transient association between HypA and HypB(AT) is controlled by the ATP hydrolysis cycle of HypB(AT), which is accelerated by HypA. Only the ATP-bound form of HypB(AT) can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB(AT) complex has a nanomolar affinity (K-d = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 mu M) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB(AT), affecting its association with HypA. These findings indicate that HypA and HypB(AT) constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB(AT) functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

DOI： 10.1073/pnas.1503102112

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.

DOI： 10.1242/jcs.153239

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1 alpha (Ero1 alpha), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1 alpha-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1 alpha; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding beta-hairpin of Ero1 alpha, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1 alpha and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.

DOI： 10.1074/jbc.M114.564104

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER.

DOI： 10.1016/j.str.2013.12.013

記述言語：英語   掲載種別：研究論文（学術雑誌）  

7800

DOI： 10.1371/journal.pone.0081440

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2013.09.063

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Structural basis of disulfide bond formation in the bacterial periplasm and ER.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44's active cysteine, simultaneously unmask the substrate binding site and -RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles. © 2013 Elsevier Inc.

DOI： 10.1016/j.molcel.2013.04.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

7804

記述言語：英語  

Almost all organisms, from bacteria to humans, possess catalytic systems that promote disulfide bond formation-coupled protein folding, i.e. oxidative protein folding. These systems are necessary for the biosynthesis of many secretory and membrane proteins, such as antibodies, major histocompatibility complex molecules, growth factors, and insulin. Over the last decade, structural studies have made striking progress in this field of research, identifying how oxidative systems operate in a specific and regulated manner to maintain redox and protein homeostasis within cells. Interestingly, more and more novel catalysts that promote disulfide bond formation have been discovered in mammals, suggesting that the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought. This review highlights the physiological roles and molecular bases of the disulfide bond formation pathways that have evolved in the bacterial periplasm and the endoplasmic reticulum of fungi and mammals. Accumulating knowledge about disulfide bond formation networks widely distributed throughout the biological kingdom has significantly advanced our understanding of the cellular mechanisms dedicated to protein quality control.

DOI： 10.1111/j.1742-4658.2012.08593.x

記述言語：英語  

Significance: Disulfide bond formation is an essential reaction involved in the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells utilize various disulfide oxidoreductases and redox-active cofactors to accelerate this oxidative reaction, and higher eukaryotes have diversified and refined these disulfide-introducing cascades over the course of evolution. Recent Advances: In the past decade, atomic resolution structures have been solved for an increasing number of disulfide oxidoreductases, thereby revealing the structural and mechanistic basis of cellular disulfide bond formation systems. Critical Issues: In this review, we focus on the evolution, structure, and regulatory mechanisms of endoplasmic reticulum oxidoreductin 1 (Ero1) family enzymes, the primary disulfide bond-generating catalysts in the endoplasmic reticulum (ER). Detailed comparison of Ero1 with other oxidoreductases, such as Prx4, QSOX, Erv1/2, and disulfide bond protein B (DsbB), provides important insight into how this ER-resident flavoenzyme acts in a regulated and specific manner to maintain redox and protein homeostasis in eukaryotic cells. Future Directions: Currently, it is presumed that multiple pathways in addition to that mediated by Ero1 cooperate to achieve oxidative folding of many secretory and membrane proteins in mammalian cells. The important open question is how each oxidative pathway works distinctly or redundantly in response to various cellular conditions. Antioxid. Redox Signal. 16, 790-799.

DOI： 10.1089/ars.2011.4418

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Molecular Bases of Cyclic and Specific Disulfide Interchange between Human ERO1 alpha Protein and Protein-disulfide Isomerase (PDI)
In the endoplasmic reticulum (ER) of human cells, ERO1 alpha and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1 alpha is essential to avoid ER hyperoxidation. To investigate how ERO1 alpha oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding beta-hairpin of ERO1 alpha specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1 alpha associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1 alpha to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1 alpha with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1 alpha-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1 alpha activity may suggest its physiological role in ER redox and protein homeostasis.

DOI： 10.1074/jbc.M111.231357

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.

DOI： 10.1016/j.molcel.2011.01.021

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nemo-like kinase (NLK) is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. Here we show that homodimerization of NLK is required for its activation and nuclear localization. Biochemical analysis revealed that NLK is activated through intermolecular autophosphorylation of NLK dimers at Thr-286. Mutation of NLK at Cys-425, which corresponds to the defect in the Caenorhabditis elegans NLK homologue lit-1, prevented NLK dimerization, rendering NLK defective in both nuclear localization and kinase activity. By contrast, the external addition of nerve growth factor, which has been previously identified as an NLK activator, induced dimerization and Thr-286 autophosphorylation of endogenous NLK proteins. In addition, both dimerization and Thr-286 phosphorylation of NLK were found to be essential for induction of neurite-like cellular processes by NLK. The present findings suggest that dimerization is an initial key event required for the functional activation of NLK.

DOI： 10.1091/mbc.E10-07-0605

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Crystal structures of human Ero1 alpha reveal the mechanisms of regulated and targeted oxidation of PDI
In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1 alpha activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1 alpha were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1 alpha modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine-tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1 alpha with the PDI b'-domain through its substrate-binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells. The EMBO Journal (2010) 29, 3330-3343. doi:10.1038/emboj.2010.222; Published online 10 September 2010

DOI： 10.1038/emboj.2010.222

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The formation of protein disulfide bonds is an oxidative reaction that is crucial for the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells possess various disulfide oxidoreductases and redox-active cofactors to accelerate this oxidative reaction in a correct manner. Crystal or solution structures have been solved for some of the oxidoreductases in the past 10 years, leading to remarkable progress in the field of thiol-based redox cell biology. Consequently, structural and mechanistic similarities in the disulfide bond formation pathways have been uncovered. This review highlights the molecular basis of the elaborate oxidative systems operating in the Escherichia coli periplasm, the endoplasmic reticulum lumen and the mitochondrial intermembrane space. The accumulated knowledge provides important insights into how protein and redox homeostasis are maintained in the cell.

DOI： 10.1111/j.1365-2443.2010.01434.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b' and a' domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a' domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a' domain influences the dynamic properties of the W domain. Moreover, the SAXS profiles revealed that oxidation of the a' active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a' domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a "closed" form releasing the oxidized substrate. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2009.11.049

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The biological kingdoms have evolved elaborate systems that ensure the catalysis of protein disulfide bond (dsb) formation in the cell. Coexisting in the periplasm of Escherichia coli are the DsbADsbB disulfide-introducing and DsbCDsbD disulfide-isomerizing pathways, which promote the oxidative folding of secreted proteins. Recent structural studies of DsbB have illuminated conformational dynamics involved in the effective oxidation of the extremely reduction-prone oxidase, DsbA, as well as the structure of the reaction centre involved in protein Dsb formation de novo in conjunction with ubiquinone. Extensive genetic and biochemical analysis has recently provided insight into how DsbD transports electrons from cytosolic thioredoxin to periplasmic DsbC. To a great extent, the molecular mechanisms of the Dsb enzyme system in E. coli have been elucidated, and are applicable to the study of protein disulfide formation systems in other organisms.

DOI： 10.1093/jb/mvp102

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104-Cys130 initial-state disulphide that is directly donated to DsbA. Here, we report the 3.4 angstrom crystal structure of a DsbB-Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB-DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41-Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane-parallel short alpha-helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130-containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.

DOI： 10.1038/emboj.2009.21

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The sigma(E) pathway of extracytoplasmic stress responses in Escherichia coli is activated through sequential cleavages of the anti-sigma(E) protein, RseA, by membrane proteases DegS and RseP. Without the first cleavage by DegS, RseP is unable to cleave full-length RseA. We previously showed that a PDZ-like domain in the RseP periplasmic region is essential for this negative regulation of RseP. We now isolated additional deregulated RseP mutants. Many of the mutations affected a periplasmic region that is N-terminal to the previously defined PDZ domain. We expressed these regions and determined their crystal structures. Consistent with a recent prediction, our results indicate that RseP has tandem, circularly permutated PDZ domains (PDZ-N and PDZ-C). Strikingly, almost all the strong mutations have been mapped around the ligand binding cleft region in PDZ-N. These results together with those of an in vitro reaction reproducing the two-step RseA cleavage suggest that the proteolytic function of RseP is controlled by ligand binding to PDZ-N.

DOI： 10.1074/jbc.M806603200

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD. In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domain and other regulatory domain lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one. Molecular details of these remarkable biochemical cascades are being rapidly unraveled by genetic, biochemical, and structural analyses in recent years.

DOI： 10.1016/j.sbi.2008.02.002

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB-DsbA ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA-DsbB oxidative and the DsbC DsbD reductive pathways has also been proposed.

DOI： 10.1107/S090904950706061X

記述言語：英語  

Structure and mechanisms of the DsbB-DsbA disulfide bond generation machine.
All organisms possess specific cellular machinery that introduces disulfide bonds into proteins newly synthesized and transported out of the cytosol. In E. coli, the membrane-integrated DsbB protein cooperates with ubiquinone to generate a disulfide bond, which is transferred to DsbA, a periplasmic dithiol oxido-reductase that serves as the direct disulfide bond donor to proteins folding oxidatively in this compartment. Despite the extensive accumulation of knowledge on this oxidation system, molecular details of the DsbB reaction mechanisms had been controversial due partly to the lack of structural information until our recent determination of the crystal structure of a DsbA-DsbB-ubiquinone complex. In this review we discuss the structural and chemical nature of reaction intermediates in the DsbB catalysis and the illuminated molecular mechanisms that account for the de novo formation of a disulfide bond and its donation to DsbA. It is suggested that DsbB gains the ability to oxidize its specific substrate, DsbA, having very high redox potential, by undergoing a DsbA-induced rearrangement of cysteine residues. One of the DsbB cysteines that are now reduced then interacts with ubiquinone to form a charge transfer complex, leading to the regeneration of a disulfide at the DsbB active site, and the cycle can begin anew. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2007.11.006

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 angstrom resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.

DOI： 10.1016/j.cell.2006.10.034

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DsbB, an Escherichia coli plasma membrane protein, oxidizes DsbA, the protein dithiol oxidant in the periplasm, in conjunction with respiratory quinone molecules. While its two periplasmic regions, in particular the essential Cys41-Cys44 and the Cys104-Cys130 cysteine pairs, have been characterized in considerable detail, little or no information is available about the functional importance of its three cytosolically disposed regions. In this work the authors introduced insertion and substitution mutations into the short (similar to 6 residue) central cytosolic loop. The purified mutant proteins proved to have two of the essential cysteines reduced and to exhibit the spectroscopic transition of bound ubiquinone constitutively. A thrombin-cleavage site present in a mutant protein called DsbB-T established that the mutant protein had a rearranged Cys41-Cys130 disulfide that would unpair Cys44. Although this covalent structure of DsbB is reminiscent of the DsbB-DsbA intermediate, in which unpaired Cys44 induces the ubiquinone transition, it is inactive because of the premature disulfide rearrangement without involving DsbA. In addition, ubiquione-mediated in vitro oxidation of reduced DsbB-T was aborted at a half-oxidized state, without rapidly producing the fully oxidized enzyme. Thus, the cytosolic loop alterations compromised the catalytic turnover of DsbB in vitro. These observations suggest that the cytosolic loop is important to coordinate the active-site residues of DsbB and ubiquinone to allow their proper reaction cycles.

DOI： 10.1089/ars.2006.8.743

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created de novo. DsbB, an Escherichia coli plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink (lambda(max) approximate to 500 nm, ubiquinone) or violet (lambda(max)approximate to 550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the a-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanicliniurn moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at -500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adcluctthat accepts a nucleophilic attack from another cysteine to generate a disulficle bond de novo. A similar mechanism is conceivable for a class of eukaryotic dithiol oxiclases having a FAD cofactor.

DOI： 10.1073/pnas.0507570103

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DsbB is a disulfide oxidoreductase present in the Escherichia coli plasma membrane. Its cysteine pairs, Cys(41)-Cys(44) and Cys(104)-Cys(130), facing the periplasm, as well as the bound quinone molecules play crucial roles in oxidizing DsbA, the protein dithiol oxidant in the periplasm. In this study, we characterized quinone-free forms of DsbB prepared from mutant cells unable to synthesize ubiquinone and menaquinone. While such preparations lacked detectable quinones, previously reported lauroylsarcosine treatment was ineffective in removing DsbB-associated quinones. Moreover, DsbB-bound quinone was shown to contribute to the redox-dependent fluorescence changes observed with DsbB. Now we reconfirmed that redox potentials of cysteine pairs of quinone-free DsbB are lower than that of DsbA, as far as determined in dithiothreitol redox buffer. Nevertheless, the quinone-free DsbB was able to oxidize similar to 40% of DsbA in a 1:1 stoichiometric reaction, in which hemi-oxidized forms of DsbB having either disulfide are generated. It was suggested that the DsbB-DsbA system is designed in such a way that specific interaction of the two components enables the thiol-disulfide exchanges in the "forward" direction. In addition, a minor fraction of quinone-free DsbB formed the DsbA-DsbB disulfide complex stably. Our results show that the rapid and the slow pathways of DsbA oxidation can proceed up to significant points, after which these reactions must be completed and recycled by quinones under physiological conditions. We discuss the significance of having such multiple reaction pathways for the DsbB-dependent DsbA oxidation.

DOI： 10.1074/jbc.M506189200

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys(41) and Cys(44) of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambda(max) = 550 nm), which required a reduced state of Cys(44) as shown previously for UQ color development (lambda(max) = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degreesC, two reaction components were observed, one completing within minutes and the other taking > 1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB( MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.

DOI： 10.1074/jbc.M407153200

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys(30)-Pro-His-Cys(33) active site of DsbA, the primary dithiol oxidant in the periplasm. Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule. This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color. The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB. Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood Of Cys(44). We propose that the Cys(44)-induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.

DOI： 10.1074/jbc.M310765200

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein disulfide bond formation in the bacterial periplasm is catalyzed by the Dsb enzymes in conjunction with the respiratory quinone components. Here we characterized redox properties of the redox active sites in DsbB to gain further insights into the catalytic mechanisms of DsbA oxidation. The standard redox potential of DsbB was determined to be -0.21 V for Cys41/Cys44 in the N-terminal periplasmic region (P1) and -0.25 V for Cys104/Cys130 in the C-terminal periplasmic region (P2), while that of Cys30/Cys33 in DsbA was -0.12 V. To our surprise, DsbB, an oxidant for DsbA, is intrinsically more reducing than DsbA. Ubiquinone anomalously affected the apparent redox property of the P1 domain, and mutational alterations of the P1 region significantly lowered the catalytic turnover. It is inferred that ubiquinone, a high redox potential compound, drives the electron flow by interacting with the P1 region with the Cys41/Cys44 active site. Thus, DsbB can mediate electron flow from DsbA to ubiquinone irrespective of the intrinsic redox potential of the Cys residues involved.

DOI： 10.1093/emboj/21.11.2646

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chymotrypsin inhibitor 2 (CI2) is the archetypal single-foldon protein that folds in simple two-state kinetics without the accumulation of a folding intermediate. To model the effects of fusion of single foldons to give a multi-foldon protein, we engineered a "double-CI2" protein, in which another CI2 polypeptide was inserted into the loop region of the parent CI2. CD and HSQC spectra demonstrated that while the double-CI2 protein adopted two kinds of native conformations, CI2-like structure was almost preserved in both the domains of double-CI2. In the folding kinetic studies, double-CI2 exhibited a remarkable rollover of the observed folding rates at low denaturant concentrations, indicating that double-CI2 accumulated a kinetic folding intermediate. The different folding mechanisms between WT-CI2 and double-CI2 support the present view that protein size or number of domains is an important determinant for formation of folding intermediates. (C) 2000 Academic Press.

DOI： 10.1006/jmbi.2000.4024

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mel. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the beta alpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin a-subunit. Based on the gel chromatography, the beta alpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[beta alpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[beta alpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl NdeltaH proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[beta alpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the beta alpha(HBM)-subunit in tetrameric alpha(2)[beta alpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits, The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins, Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.

DOI： 10.1074/jbc.275.17.12438

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The crystal structure of the homotetramer of a chimera beta alpha-subunit of human hemoglobin was refined at 2.5 Angstrom resolution. The chimera subunit was constructed by replacing an exon-encoded module M4 of the beta-subunit with that of the alpha-subunit, simulating an exon-shuffling event. The implanted module M4 retained the native alpha-subunit structure, while module M3 was disturbed around the site where a new type of intron was recently found. Some of the residues were found in alternative conformations that avoid steric hindrance at the subunit interface. The modules are modestly rigid in their backbone structures by using side-chains to compensate for interface incompatibility. (C) 1999 Academic Press.

DOI： 10.1006/jmbi.1999.2603

記述言語：英語   掲載種別：研究論文（学術雑誌）  

To investigate structural and functional significance of a newly proposed structural unit in globins, the "heme binding module", we synthesized a "heme binding module"-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin alpha-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin alpha-subunit converted the absorption spectra into that of the alpha-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the alpha-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins. (C) 1998 Academic Press.

DOI： 10.1006/jmbi.1998.2073

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A chimera beta alpha-subunit of human hemoglobin was crystallized into a carbon-monoxy form, The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Angstrom. The crystal belongs to the monoclinic space group P2(1), with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Angstrom, and beta = 91.0 degrees. These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263-267, 1998. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0134(19980815)32:3<263::AID-PROT1>3.0.CO;2-J

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Functional and structural significance of the "module" in proteins has been investigated for globin proteins. Our previous studies have revealed that some modules in globins are responsible for regulating the subunit association and heme environmental structures, whereas the module substitution often induces fatal structural destabilization, resulting in failure of functional regulation. In this paper, to gain further insight into functional and structural significance of the modular structure in globins, we focused upon the "pseudo-module" in globin structure where boundaries are located at the center of modules. Although the pseudo-module has been supposed not to retain a compactness, the beta alpha(PM3)-subunit, in which one of the pseudo-modules, the F1-H6 regoin, of the alpha-subunit is implanted into the beta-subunit, conserved stable globin structure, and its association property was converted into that of the alpha-subunit, as the case for the module substituted globin, the beta alpha(M4)-subunit. These results suggest that modules are not unique structural and functional units for globins. Interestingly, however, the recent reconsideration of the module boundary indicates that the modules in globins can be further divided into two small modules, and one of the boundaries for the new small modules coincides with that of the pseudo-module we substituted in this study. Although it would be premature to conclude the significance of the modular structure in globins, it can be safely said that we have found new structural units in globin structure, probably new modules.

DOI： 10.1074/jbc.273.14.8080

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The alpha- and beta-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric alpha alpha beta- and beta beta alpha-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric alpha alpha beta-subunit drastically collapsed, while the chimeric beta beta alpha-subunit conserved the native globin structure (Wakasugi, K., Ishimori, It., Imai, IT., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric alpha alpha beta-subunit. Based on tryptophan-fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp(14) and Tyr(125) would be induced in the chimeric alpha alpha beta-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure, On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the beta beta alpha-subunit The different stabilities of these module-substituted globins imply that nodules would not always be stable ''structural'' units, and interactions between modules are crucial to construct stable globin subunits.

DOI： 10.1074/jbc.272.48.30054

開催年月日： 2019年7月

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開催年月日： 2019年5月

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開催年月日： 2019年5月

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開催年月日： 2018年9月

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開催年月日： 2018年9月

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Structural basis of pH- and zinc-dependent multiple client recognition by ERp44

開催年月日： 2018年9月

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開催年月日： 2018年8月

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開催年月日： 2018年8月

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Cooperation of pH and zinc ions in ERp44-mediated protein quality control at the ER-Golgi interface

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Crystal Structure of DsbB-DsbA Complex Revealing a Cysteine Relocation Mechanism

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Crystal structure of the DsbB-DsbA complex revealing a cysteine relocation process for disulfide bond generation

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Crystal Structure of DsbB-DsbA Complex Revealing a Cysteine Relocation Mechanism

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How are protein disulfide bonds generated in the cell?

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Structural basis of the DsbA-DsbB-UQ oxidative system in E. coli.

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Crystal structure of the DsbB-DsbA complex reveals a mechanism of protein disulfide bond generation in E. coli.

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Structural basis for protein disulfide generation in the cell

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Structure and mechanism of the DsbB-DsbA protein disulfide generation system in E. coli

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Crystal structures of mammalian human Ero1 and ERdj5, the enzymes involved in ER quality control

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Structural basis of regulated protein disulfide bond formation in human cells

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Structure and mechanism of protein disulfide bond formation systems in human cells

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Structural and mechanistic basis of regulated and specific protein disulfide bond formation in human cells

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Structural and mechanistic basis of protein disulfide bond formation network in mammalian cells

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Structural and mechanistic basis of the protein disulfide bond formation system in mammalian cells

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Structural and mechanistic basis of the protein disulfide bond formation system in mammalian cells

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The protein disulfide bond formation system in mammalian cells

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Dual conformations of ERdj5 play a significant role in acceleration of the ER associated degradation.

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Dynamic natures of PDI family members in catalysis of oxidative protein folding and ER associated degradation

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Dynamic natures of PDI family members in catalysis of oxidative protein folding and ER associated degradation

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How do PDI family member proteins act on unfolded/misfolded proteins to ensure protein quality control in the ER?

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The highly dynamic nature of ERdj5 plays a key role in efficient ER-associated degradation of aberrant protein oligomers

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Zinc ions regulate ERp44-mediated protein quality control in the early secretory pathway

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Zinc ions have a new physiological role in protein quality control in the early secretory pathway

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Novel protein quality control system cooperated by zinc ions and ERp44 in the early secretory pathway

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Structural basis for redox-regulated galectin1 function

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Understanding the folding pathways of proinsulin

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Impact of Ca2+ on the function interplay between ERp57 and Calnexin

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Molecular basis of redox-, pH-, and metal ion-dependent protein quality control at the ER-Golgi interface

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Protein Disulfide Isomerase family; their molecular actions and functions

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Molecular basis of redox- and zinc-dependent protein quality control at the ER-Golgi interface

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Close linkage between redox and zinc homeostasis in the endoplasmic reticulum

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Molecular basis of zinc-dependent protein quality control at the ER-Golgi interface

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Cryo-EM Structures of Human Zinc Transporter ZnT7 Reveal the Mechanism of Zn2+ Uptake into the Golgi Apparatus

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Mechanisms of calcium homeostasis in the ER and Golgi apparatus revealed by cross-scale microscopic measurements.

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Structure analysis of a cargo receptor ERGIC-53 homolog, ERGL

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Structural analysis of cargo transport by ERGIC-53

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Insulin quality control mechanism in the endoplasmic reticulum

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Zinc homeostasis and proteostasis at the ER-Golgi interface

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Yuta Amagai, Kenji Inaba

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The brave to change your research theme: let's establish our own research field!

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次世代のアカデミア創薬を担う若手の力 抗がん剤を目指した若手研究者連携による創薬スクリーニング

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膵臓で特異的に発現するPDIファミリータンパク質(PDIp)の生理的機能の解析

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小胞体ストレスタンパク質を標的とした抗癌剤スクリーニング

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膵特異的に発現するPDIファミリータンパク質(PDIp)の生理的機能の解析

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[NiFe]ヒドロゲナーゼへNiを組み込むHypAB複合体の結晶構造解析

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新規PDIファミリータンパク質ERp46の構造と機能

DOI： 10.2142/biophys.55.034

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ERdj5(JPDI)基質候補タンパク質のマウス個体組織からの網羅的同定

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哺乳動物細胞の小胞体におけるジスルフィド結合形成ネットワークの構造基盤

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哺乳動物細胞小胞体に局在するERdj5(JPDI)の生理的基質候補タンパク質の網羅的解析

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分泌タンパク質の成熟化を監視するpHに依存的な新たなタンパク質品質管理機構の発見

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1P009 NMR analyses of relation-ship between multidomain structure and functions of protein disulfide isomerase

DOI： 10.2142/biophys.44.S32_1

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Three-dimensional structure and domain-domain interactions of protein disulfide isomerase as revealed by NMR spectroscopy

DOI： 10.2142/biophys.43.S45_4

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2R1600 Structural analyses of protein disulfide isomerase focused on the active sites and domain-domain interactions

DOI： 10.2142/biophys.42.S150_3

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Structure of the folding intermediate of apomyolobin studied by fluorescence quenching.

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Effects of intersubunit hydrogen bonds on association property of hemoglobin

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Crystal structure analyses of module substituted chimera hemoglobins

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Structures and Functions of the artificial globins designed by the substitution of the heme binding module

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