Thanh Do Tuan, Motonori Matsusaki, Honoka Ota, Soichiro Kawagoe, Dilini Hettimudalige Nisansala, Munehiro Kumashiro, Noriyoshi Isozumi, Hiroyuki Kumeta, Yohei Kono, Takeshi Shimi, Koichiro Ishimori, Eiichiro Mori, Satoshi Arai and Tomohide Saio : OptoChaperone A Biohybrid Tool for Regulating Protein Condensates in Cells and In Vitro, Journal of the American Chemical Society, 148, 16, 17429-17442, 2026.
(Summary)
Protein condensates formed via liquid-liquid phase separation (LLPS) are increasingly recognized as key players in diverse cellular processes, including those associated with disease. Despite extensive efforts to characterize their formation and function, tools that enable precise, reversible, and spatiotemporal control of LLPS remain limited. Here, we report OptoChaperone, a light-activatable molecular system designed to manipulate protein condensates both in vitro and in living cells. This biohybrid system leverages photoresponsive switching to control chaperone activity: blue light triggers the suppressive function, leading to the dissolution of protein condensates, whereas UV light deactivates the system, allowing condensate formation. We demonstrate the efficacy of OptoChaperone in regulating several disease-related protein condensates, such as fused in sarcoma, TAR DNA-binding protein 43, and heat shock factor 1. Importantly, the system exhibits reversible and robust control over droplet dynamics without requiring chemical additives or genetic modifications of the client proteins. Given the reversibility and efficiency of OptoChaperone in the manipulation of protein condensates, this tool offers a powerful platform for dissecting the roles of protein condensation in cellular physiology and pathology. This strategy also holds potential for broader applications in synthetic biology, biomolecular engineering, and therapeutic modulation of aberrant phase separation.
The endoplasmic reticulum (ER) plays crucial roles in maintaining protein quality control and regulating dynamic Ca2+ storage in eukaryotic cells. However, the proteostasis system involved in ER-mediated protein quality control has not been fully characterized. Here we show that Ca2+ triggers the condensation of PDIA6, an ER-resident disulfide isomerase and molecular chaperone, into quality control granules. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the PDIA6 condensates recruit proinsulin, thereby accelerating the oxidative proinsulin folding and suppressing the proinsulin aggregation inside quality control granules, essential for secretion of insulin.
Hanif Ali, Mone Yamanishi, Miki Tsuchiya, Rumana Yesmin Hasi, Motonori Matsusaki, Tomohide Saio, Mutsumi Aihara, Ryushi Kawakami and Tamotsu Tanaka : Solubilization of saturated fatty acids and its lysophosphatidylcholine by complexation with bovine serum albumin, Lipids, 61, 19-28, 2025.
(Summary)
High plasma levels of saturated fatty acids (SFAs) are associated with lifestyle diseases such as atherosclerosis and diabetes, whereas the accumulation of very long-chain fatty acids (VLCFAs) is believed to be responsible for neuropathy in certain types of peroxisomal disorders. Despite their clinical relevance, the toxicity mechanisms of fatty acids (FAs) remain poorly understood, largely because of the challenges in solubilizing them for in vitro experiments. We recently developed a method to form stable complexes of FAs with bovine serum albumin (BSA) using isopropanol as the solvent. Here, we demonstrate the stability and concentration range of FA/BSA and lysophosphatidylcholine (LysoPtdCho)/BSA complexes prepared using this method. These complexes exhibit enhanced solubility, retain their biological activity in cellular uptake assays, and remain stable for up to 12 months. We believe that our method will contribute to a better understanding of the toxicity and metabolism of SFAs and SFA-LysoPtdChos, and offer new insights into their roles in metabolic diseases and peroxisomal disorders.
Keita Mori, Tsubura Kuramochi, Motonori Matsusaki, Yuki Hashiguchi, Masaki Okumura, Tomohide Saio, Yoshiaki Furukawa, Kenta Arai and Takahiro Muraoka : Metal-Responsive Up-Regulation of Bifunctional Disulfides for Suppressing Protein Misfolding and Promoting Oxidative Folding, Angewandte Chemie International Edition, 64, 36, e202502187, 2025.
(Summary)
The stress-responsive up-regulation process is a sophisticated biological response to maintain cellular homeostasis. In intracellular anti-oxidant systems, the expression level of oxidoreductases is up-regulated under oxidative stress, mitigating oxidative damage on biomolecules and enhancing protein folding capacity. Herein, inspired by the biological system, we developed a synthetic folding promotor whose reactivity is up-regulated under stress conditions. We conjugated two metal-binding 1,4,7,11-tetraazacyclotetradecane (cyclam) ligands and a redox-active disulfide to obtain cyclam-SS, whose reactivity can be enhanced under metal-induced stress. Metal coordination increased the redox potential of cyclam-SS, activating it as an oxidant. While CuII ions severely hampered the oxidative folding of substrate polypeptides, cyclam-SS exhibited bifunctional folding-promoting properties, i) suppressing CuII-mediated misfolding and aggregation, and ii) harnessing CuII to enhance oxidative folding. Cyclam-SS was also useful for disulfide-bond formation to promote oxidative folding of pharmaceutical and pathological proteins, as demonstrated with proinsulin and superoxide dismutase 1 (SOD1). Furthermore, cyclam-SS protected cultured cells from copper-induced stress. Thus, we demonstrated the induction of the stress-responsive up-regulation process by a bifunctional folding promotor controlling the folding status of biologically important proteins under metal-induced stress. The strategy of "stress-responsive up-regulation" could aid the development of novel synthetic materials for treating intracellular stress and related disorders.
Motonori Matsusaki, Rina Okada, Yuya Tanikawa, Shingo Kanemura, Dai Ito, Yuxi Lin, Mai Watabe, Hiroshi Yamaguchi, Tomohide Saio, Young-Ho Lee, Kenji Inaba and Masaki Okumura : Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding, Biology, 10, 11, 1112, 2021.
(Summary)
The physiological functions of proteins are destined by their unique three-dimensional structures. Almost all biological kingdoms share conserved disulfide-catalysts and chaperone networks that assist in correct protein folding and prevent aggregation. Disruption of these networks is implicated in pathogenesis, including neurodegenerative disease. In the mammalian endoplasmic reticulum (ER), more than 20 members of the protein disulfide isomerase family (PDIs) are believed to cooperate in the client folding pathway, but it remains unclear whether complex formation among PDIs via non-covalent interaction is involved in regulating their enzymatic and chaperone functions. Herein, we report novel functional hetero complexes between PDIs that promote oxidative folding and inhibit aggregation along client folding. The findings provide insight into the physiological significance of disulfide-catalyst and chaperone networks and clues for understanding pathogenesis associated with disruption of the networks.
(Keyword)
protein disulfide isomerase family / disulfide bond / endoplasmic reticulum / oxidative folding / molecular chaperone / protein-protein interaction
Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment.
Yuya Tanikawa, Shingo Kanemura, Dai Ito, Yuxi Lin, Motonori Matsusaki, Kimiko Kuroki, Hiroshi Yamaguchi, Katsumi Maenaka, Young-Ho Lee, Kenji Inaba and Masaki Okumura : Ca Regulates ERp57-Calnexin Complex Formation., Molecules, 26, 10, 2853, 2021.
(Summary)
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 Ca 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 Ca. 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 Ca depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca.
(Keyword)
Calcium / Calnexin / Disulfides / Humans / Models, Biological / Oxidation-Reduction / Protein Aggregates / Protein Binding / Protein Disulfide-Isomerases / Protein Folding / Thermodynamics
Masaki Okumura, Shingo Kanemura, Motonori Matsusaki, Misaki Kinoshita, Tomohide Saio, Dai Ito, Chihiro Hirayama, Hiroyuki Kumeta, Mai Watabe, Yuta Amagai, Young-Ho Lee, Shuji Akiyama and Kenji Inaba : A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization., Structure, 29, 12, 1357-1370.E6, 2021.
(Summary)
P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on 30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.
Shunsuke Okada, Motonori Matsusaki, Masaki Okumura and Takahiro Muraoka : Conjugate of Thiol and Guanidyl Units with Oligoethylene Glycol Linkage for Manipulation of Oxidative Protein Folding., Molecules, 26, 4, 879, 2021.
(Summary)
Oxidative protein folding is a biological process to obtain a native conformation of a protein through disulfide-bond formation between cysteine residues. In a cell, disulfide-catalysts such as protein disulfide isomerase promote the oxidative protein folding. Inspired by the active sites of the disulfide-catalysts, synthetic redox-active thiol compounds have been developed, which have shown significant promotion of the folding processes. In our previous study, coupling effects of a thiol group and guanidyl unit on the folding promotion were reported. Herein, we investigated the influences of a spacer between the thiol group and guanidyl unit. A conjugate between thiol and guanidyl units with a diethylene glycol spacer (GdnDEG-SH) showed lower folding promotion effect compared to the thiol-guanidyl conjugate without the spacer (GdnSH). Lower acidity and a more reductive property of the thiol group of GdnDEG-SH compared to those of GdnSH likely resulted in the reduced efficiency of the folding promotion. Thus, the spacer between the thiol and guanidyl groups is critical for the promotion of oxidative protein folding.
Shingo Kanemura, Motonori Matsusaki, Kenji Inaba and Masaki Okumura : PDI Family Members as Guides for Client Folding and Assembly., International Journal of Molecular Sciences, 21, 24, 9351, 2020.
(Summary)
Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.
(Keyword)
Animals / Calnexin / Calreticulin / Humans / Protein Disulfide-Isomerases / Protein Folding / Protein Multimerization / Proteostasis
Aya Okuda, Motonori Matsusaki, Taro Masuda, Ken Morishima, Nobuhiro Sato, Rintaro Inoue, Masaaki Sugiyama and Reiko Urade : A novel soybean protein disulphide isomerase family protein possesses dithiol oxidation activity: identification and characterization of GmPDIL6., The Journal of Biochemistry, 168, 4, 393-405, 2020.
(Summary)
Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulphide bonds, viz. oxidative folding, catalysed by the protein disulphide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active centre (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulphide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was up-regulated under ER stress. GmPDIL6 may play a role in the formation of disulphide bonds in nascent proteins for oxidative folding in the ER.
Motonori Matsusaki, Shingo Kanemura, Misaki Kinoshita, Young-Ho Lee, Kenji Inaba and Masaki Okumura : The Protein Disulfide Isomerase Family: from proteostasis to pathogenesis., Biochimica et Biophysica Acta (BBA) - General Subjects, 1864, 2, 129338, 2019.
(Summary)
In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by approximately 20 members of the protein disulfide isomerase family (PDIs). PDIs consist of multiple thioredoxin-like domains and recognize a wide variety of proteins via highly conserved interdomain flexibility. Although PDIs have been studied intensely for almost 50 years, exactly how they maintain protein homeostasis in the ER remains unknown, and is important not only for fundamental biological understanding but also for protein misfolding- and aggregation-related pathophysiology. Herein, we review recent advances in structural biology and biophysical approaches that explore the underlying mechanism by which PDIs fulfil their distinct functions to promote productive protein folding and scavenge misfolded proteins in the ER, the primary factory for efficient production of the secretome.
(Keyword)
Animals / Disulfides / Endoplasmic Reticulum / Humans / Membrane Glycoproteins / Mice / Mutation / Neurodegenerative Diseases / Oxidation-Reduction / Oxidative Stress / Peptides / Protein Denaturation / Protein Disulfide-Isomerases / Protein Domains / Protein Folding / Rats
Motonori Matsusaki : Protein Function Depending on the 'Distribution' of Assembly and 3D Conformation, Expert Symposium on Marine Genetic Resource Utilization,
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Motonori Matsusaki and Tomohide Saio : Advancing native electrophoresis through fluorescence detection and automated image analysis for protein conformational dynamics and assembly, 2025 Korea-Japan International Symposium on Protein Science, Himeji, Jun. 2025.
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Motonori Matsusaki, Tomohide Saio, Inaba Kenji and Okumura Masaki : Higher-order Oligomerization of IRE1 Luminal Domain Modulates Unfolded Protein Response, 16th ER and Redox club meeting, Namur, Mar. 2025.
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Motonori Matsusaki and Tomohide Saio : High-resolution visualization of heterogenous protein assembly, Korea-Japan International Symposium on Protein Science (Joint Conference), Sapporo, Jun. 2024.
Proceeding of Domestic Conference:
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Yuki Kuwano, Keyoumu Nazere, Motonori Matsusaki, Takemura Ren, Ryosuke Miyamoto, Tomohide Saio and Hiroyuki Morino : Epitranscriptomic control of FUS condensation:m6A as a druggable regulator of ALS-related pathology, 第67回日本神経学会学術大会, May 2026.
Tuan Thanh Do, Motonori Matsusaki, Soichiro Kawagoe, Kumeta Hiroyuki and Tomohide Saio : OptoChaperone-A Biohybrid Tool Regulating Protein Condensates in vitro and in Cells, 第63回日本生物物理学会年会, Sep. 2025.
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Motonori Matsusaki and Tomohide Saio : Decoding Protein Assembly and Its Dynamic State Distributions with Native Electrophoresis, 第 25 回日本蛋白質科学会年会, Jun. 2025.