掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-025-57659-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

According to classical Anfinsen's dogma, a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, certain proteins may fold into two or more conformations from single amino acid sequences. Here, we designed a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal β-strand and dihydrofolate reductase (DHFR) lacking the N-terminal β-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analyses, including atomic force microscopy (AFM) imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. The drastic conformational change upon the ligand binding was directly visualized by high-speed AFM. Furthermore, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a unique direction for creating artificial proteins with on-off behaviors.

DOI： 10.1073/pnas.2412117122

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-025-55853-7

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その他リンク： https://www.nature.com/articles/s41467-025-55853-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We demonstrated a novel approach for protein-protein interaction (PPI) profiling of histone H3 using intracellular photocatalytic-proximity labeling (iPPL). This approach identified that the combination of acriflavine as a photocatalyst and 1-methyl-4-arylurazol (MAUra) as a protein labeling agent was the most efficient strategy to proceed the protein proximity labeling reaction. Furthermore, the identification of the labeled amino acids in histone H3 interacting proteins, histone lysine N-methyltransferase EZH2, showed that the amino acid in EZH2 within a few nanometers from histone H3 is labeled by iPPL. This restricted labeling radius allows for more-focused PPI profiling, compared to conventional proximity labeling methods.

DOI： 10.1021/acschembio.4c00680

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Our study demonstrates the exceptional efficiency of 1-methyl-4-arylurazole (MAUra) for tyrosine labeling, optimized with laccase under mild conditions, achieving a high efficiency (kcat/Km = 7.88 × 104 M-1 s-1) with minimal oxidative side reactions and selective labeling of highly exposed tyrosine sites on proteins.

DOI： 10.1039/d4cc03802a

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.ijbiomac.2024.133660

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出版者・発行元：Cold Spring Harbor Laboratory  

Abstract

Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion inC9orf72trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherinβ2 (Kapβ2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kapβ2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kapβ2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kapβ2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight intoC9orf72-ALS related to age and repeat expansion.

DOI： 10.1101/2024.07.03.601791

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.

DOI： 10.1093/nar/gkae309

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.celrep.2023.113569

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b+ F4/80- myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.

DOI： 10.1002/adbi.202300159

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.

DOI： 10.1002/psc.3536

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cell-based systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.

DOI： 10.1021/acssynbio.3c00191

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

DOI： 10.3390/ijms24097829

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Molecular chaperones are indispensable proteins that assist the folding of aggregation-prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best characterized chaperones are the Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome-wide experiments. These substrates comprise various proteins, but exhibit remarkable structural features. They include a number of α/β proteins, particularly those adopting the TIM β/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2-layer 2α4β sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE-depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common-substructure hypothesis and prediction method.

DOI： 10.1002/2211-5463.13590

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.

DOI： 10.1093/nar/gkac1276

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Escherichia coli has multiple pathways to release nonproductive ribosome complexes stalled at the 3' end of nonstop mRNA: tmRNA (SsrA RNA)-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4-57 prophage adjacent to E. coli ssrA leads to the inactivation of tmRNA and switches the primary rescue pathway from trans-translation to ArfA/RF2. This "rescue-switching" rearranges not only the proteome landscape in E. coli but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA+ cells, we found ZntR, whose mRNA is transcribed together as the upstream part of nonstop arfA mRNA. Repression of ZntR and reconstituted model genes depends on the translation of the downstream nonstop ORFs that trigger the trans-translation-coupled exonucleolytic degradation by polynucleotide phosphorylase (PNPase). Namely, our studies provide a novel example of trans-translation-dependent regulation and re-define the physiological roles of prophage excision.

DOI： 10.1111/mmi.15003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Robust translation elongation of any given amino acid sequence is required to shape proteomes. Nevertheless, nascent peptides occasionally destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains can stochastically induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, using budding yeast and a human factor-based reconstituted translation system, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in their N-terminal regions alter canonical ribosome dynamics, stochastically aborting translation. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we find many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk of failure. Indeed, a bioinformatics analysis reveals that the N-terminal regions of ORFs lack D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

DOI： 10.1038/s41467-022-35156-x

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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：Frontiers Media SA  

Co-translational protein folding is one of the central topics in molecular biology. In Escherichia coli, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both in vitro and in vivo. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living E. coli cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.

DOI： 10.3389/fmolb.2022.891128

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE.

DOI： 10.3390/molecules27123772

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The onset of neurodegenerative disorders (NDs), such as Alzheimer's disease, is associated with the accumulation of aggregates of misfolded proteins. We previously showed that chemical knockdown of ND-related aggregation-prone proteins can be achieved by proteolysis targeting chimeras (PROTACs). However, hetero-bifunctional PROTACs generally show poor permeability into the central nervous system, where NDs are located. Here, we document the conversion of one of our PROTACs into hydrophobic tags (HyTs), another class of degraders bearing hydrophobic degrons. This conversion decreases the molecular weight and the number of hydrogen bond donors/acceptors. All the developed HyTs lowered the level of mutant huntingtin, an aggregation-prone protein, with potency comparable to that of the parent PROTAC. Through IAM chromatography analysis and in vivo brain penetration assay of the HyTs, we discovered a brain-permeable HyT. Our results and mechanistic analysis indicate that conversion of protein degraders into HyTs could be a useful approach to improve their drug-like properties.

DOI： 10.1021/acsmedchemlett.1c00500

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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：Wiley  

DOI： 10.1002/cctc.202200077

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cctc.202200077

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

DOI： 10.1038/s41589-021-00951-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Intracellular photocatalytic-proximity labeling (iPPL) was developed to profile protein-protein interactions in the microenvironment of living cells. Acriflavine was found to be an efficient cell-membrane-permeable photocatalyst for introduction into the genetically HaloTag-fused protein of interest for iPPL with a radical labeling reagent, 1-methyl-4-arylurazole. iPPL was applied to the histone-associated protein H2B in HaloTag-H2B expressing HEK293FT cells. The proteins directly interacting with histones and RNA-binding proteins were selectively labeled in the intracellular environment, suggesting that the iPPL method has a smaller labeling radius (CA. 6 nm) than the BioID and APEX methods.

DOI： 10.1039/d1cc05764b

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, nascent polypeptide products bear an inherent risk of elongation abortion. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, weaken the translating ribosomal complex, causing certain nascent chain sequences to prematurely terminate translation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic and do not interrupt translation, due to two features of the nascent polypeptide. Firstly, the nascent polypeptide itself spans the exit tunnel, and secondly, its bulky amino acid residues occupy the tunnel entrance region, thereby serving as a bridge and protecting the large and small ribosomal subunits from dissociation. Thus, nascent polypeptide products have an inbuilt ability to ensure elongation continuity.

DOI： 10.15252/embj.2021108299

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

DOI： 10.1038/s41467-021-25560-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

DOI： 10.1002/1873-3468.14151

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

DOI： 10.1021/jacs.1c01626

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DOI： 10.1073/pnas.2016287118

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

DOI： 10.1093/jb/mvab014

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記述言語：日本語   出版者・発行元：日本細菌学会  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.jmb.2020.166750

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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：Elsevier BV  

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

DOI： 10.1016/j.isci.2021.102059

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

One aspect of the study of the origins of life focuses on how primitive chemistries assembled into the first cells on Earth and how these primitive cells evolved into modern cells. Membraneless droplets generated from liquid-liquid phase separation (LLPS) are one potential primitive cell-like compartment; current research in origins of life includes study of the structure, function, and evolution of such systems. However, the goal of primitive LLPS research is not simply curiosity or striving to understand one of life's biggest unanswered questions, but also the possibility to discover functions or structures useful for application in the modern day. Many applicational fields, including biotechnology, synthetic biology, and engineering, utilize similar phaseseparated structures to accomplish specific functions afforded by LLPS. Here, we briefly review LLPS applied to primitive compartment research and then present some examples of LLPS applied to biomolecule purification, drug delivery, artificial cell construction, waste and pollution management, and flavor encapsulation. Due to a significant focus on similar functions and structures, there appears to be much for origins of life researchers to learn from those working on LLPS in applicational fields, and vice versa, and we hope that such researchers can start meaningful cross-disciplinary collaborations in the future.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

DOI： 10.1039/d0cc02571b

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jacs.0c05937

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1042/BCJ20190723

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掲載種別：研究論文（学術雑誌）  

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

DOI： 10.1093/pcp/pcz056

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

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

DOI： 10.1002/pro.3624

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.3624

掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c9cc05231c

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

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

DOI： 10.1021/acs.biomac.8b01379

PubMed

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掲載種別：研究論文（学術雑誌）  

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

DOI： 10.1093/pcp/pcy163

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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 Publishing Group  

DOI： 10.1038/s41598-017-18977-5

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

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

DOI： 10.1074/jbc.ra118.005496

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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：American Chemical Society  

DOI： 10.1021/jacs.7b09892

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.molcel.2017.10.020

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nar/gkx776

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/jacs.6b07925

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.1520560113

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/1873-3468.12036

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep18025

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3389/fmicb.2015.01113

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/jacs.5b02144

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M114.620427

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M114.610444

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記述言語：日本語   出版者・発行元：The Biophysical Society of Japan General Incorporated Association  

Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. However, the relationship between the propensity to form aggregates and primary sequence and preferences of chaperones for substrates has not been understood. We comprehensively analyzed the aggregation propensity of all Escherichia coli proteins and the aggregation prevention effect of three major chaperones for aggregation-prone proteins by using a reconstituted chaperone-free translation system (PURE system). The resource obtained here can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.<br>

DOI： 10.2142/biophys.53.309

CiNii Books

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その他リンク： https://jlc.jst.go.jp/DN/JALC/10025906614?from=CiNii

記述言語：英語  

The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.

DOI： 10.1038/nchem.1681

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.1201380109

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/1752-0509-5-98

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/mabi.201000457

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：共立出版  

PubMed

CiNii Books

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.0811922106

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語   出版者・発行元：シーエムシー出版  

CiNii Books

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記述言語：日本語   出版者・発行元：バイオインダストリー協会  

CiNii Books

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