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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.

ABSTRACT 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.

Abstract 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 Cu^II ions severely hampered the oxidative folding of substrate polypeptides, cyclam‐SS exhibited bifunctional folding‐promoting properties, i) suppressing Cu^II‐mediated misfolding and aggregation, and ii) harnessing Cu^II 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.

Abstract The endoplasmic reticulum (ER) plays key roles in protein quality control^1,2and dynamic Ca²⁺storage^3,4in eukaryotic cells. However, the protein homeostasis (proteostasis) system that regulates these ER functions is still incompletely characterised. Previous study revealed the importance of oligomerization in the function PDIA1, an ER-resident disulfide isomerase and molecular chaperone, regulates oligomeric states in accordance with client folding⁵. This result suggests that at least some of the 20 members of other PDI family may undergo regulated self-assembly in order to optimally function. Here, we show that Ca²⁺triggers the phase separation of PDIA6 into liquid-like condensates. 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 Ca²⁺-driven condensation of PDIA6 recruits PDIA3 and proinsulin, thus increasing their local concentrations. This process results in the 30-fold enhancement of proinsulin folding and in the inhibition of proinsulin aggregation. Our findings shed light on a condensation-driven Ca²⁺-mediated proteostasis cascade in the ER by revealing how the efficiency of the protein folding process can be enhanced within quality control granules.

<jats:p>A promiscuous protein binder with redox activity efficiently promotes oxidative protein folding under condensed sub-millimolar conditions.</jats:p>

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.

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.

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+.

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.

<title>Abstract</title> 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 proteinGmPDIL6. 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."

Most proteins synthesized in the endoplasmic reticulum (ER) possess intramolecular and intermolecular disulfide bonds, which play an important role in the conformational stability and function of proteins. Hence, eukaryotic cells contain protein disulfide bond formation pathways such as the protein disulfide isomerase (PDI)-ER oxidoreductin 1 (Ero1) system in the ER lumen. In this study, we identified soybean PDIL7 (GmPDIL7), a novel soybean ER membrane-bound PDI family protein, and determined its enzymatic properties. GmPDIL7 has a putative N-terminal signal sequence, a thioredoxin domain with an active center motif (CGHC), and a putative C-terminal transmembrane region. Likewise, we demonstrated that GmPDIL7 is ubiquitously expressed in soybean tissues and is localized in the ER membrane. Furthermore, GmPDIL7 associated with other soybean PDI family proteins in vivo and GmPDIL7 mRNA was slightly upregulated under ER stress. The redox potential of recombinant GmPDIL7 expressed in Escherichia coli was -187 mV, indicating that GmPDIL7 could oxidize unfolded proteins. GmPDIL7 exhibited a dithiol oxidase activity level that was similar to other soybean PDI family proteins. However, the oxidative refolding activity of GmPDIL7 was lower than other soybean PDI family proteins. GmPDIL7 was well oxidized by GmERO1. Taken together, our results indicated that GmPDIL7 primarily plays a role as a supplier of disulfide bonds in nascent proteins for oxidative folding on the ER membrane. Database The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Enzyme Protein disulfide isomerase

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a' domain among the a, a', and b domains of GmPDIM. A disulfide bond introduced into the active center of the a' domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a' domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants.

Multiple enzymatic systems can catalyse protein disulfide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. The enzyme quiescin sulfhydryl oxidase (QSOX) catalyses disulfide bond formation in unfolded proteins via the reduction of oxygen. We found two QSOX homologues in the soybean genome database, Glycine max QSOX (GmQSOX) 1 and GmQSOX2, which encode proteins composed of an N-terminal signal peptide, a thioredoxin-like domain, an FAD-binding domain, Erv/ALR, and a transmembrane region near the C terminus. We subsequently cloned two GmQSOX1 cDNAs, GmQSOX1a and GmQSOX1b, which may be generated by alternative splicing. The GmQSOX1a, GmQSOX1b and GmQSOX2 mRNA levels increased during seed storage protein synthesis in the cotyledon, and were also upregulated under conditions causing ER stress. Recombinant GmQSOX1 expressed in Escherichia coli formed disulfide bonds on reduced and denatured RNase A, but did not show any refolding activity. The reduced and denatured RNase A was effectively refolded by recombinant GmQSOX1 in the presence of the soybean protein disulfide isomerase family protein GmPDIL-2 in the absence of glutathione redox buffer, suggesting that GmQSOX1 plays a role in protein folding in the ER.

beta-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean beta-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the alpha'- and alpha-subunits of beta-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the beta-conglycinin complexes containing the disulfide-linked alpha'/alpha-subunits were complexes of more than 720 kD. The alpha'- and alpha-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between alpha'/alpha-subunits residing in different beta-conglycinin hexamers, but the binding of P34 to alpha'- and alpha-subunits reduces the linkage between beta-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when beta-conglycinin was expressed under nonreducing conditions.