Oxidative protein folding, which is critical to proteins achieving their functional structures, is catalyzed in cells by protein disulfide isomerase (PDI)an enzyme that couples redox catalysis with the transient capture of folding intermediates to promote native disulfide formation while preventing aggregation. Although PDI improves oxidative folding in both chemically synthesized and recombinantly produced proteins, its use is restricted to homogeneous systems, limiting reusability and operational robustness. Artificial PDI mimics have advanced in vitro folding; however, no system has yet combined sufficient redox activity for native disulfide formation with a folding environment that suppresses aggregation, nor demonstrated true reusability. Here, we introduce a polymer-based "solid chaperone" that realizes PDI-like dual activity on an abiotic surface, achieving what natural PDI cannot: recyclable, HPLC-free oxidative folding without the stability and single-use limitations of enzymes. The covalent immobilization of cyclic diselenide onto polystyrene beads yields a redox-active and hydrophobic interface that transiently captures unfolded proteins, catalyzes both disulfide bond formation and isomerization, and suppresses aggregation even at high substrate concentrations. This solid-phase catalyst outperformed its homogeneous counterpart, producing native peptides and proteins in up to 99% yield and retaining full activity over multiple reuse cycles. These results demonstrate that complex biological folding functions, once confined to fragile enzymes, can be re-engineered into durable polymeric materials. This solid-phase strategy not only enables recyclable oxidative folding but also establishes a paradigm for translating enzymatic behavior into scalable synthetic systems with industrial potential.

Proteostasis maintains the balance between protein synthesis, folding, anddegradation within the endoplasmic reticulum (ER). This quality-control system ensures that proteins undergo proper post-translational modifications-such as PDI-ERO1-mediated oxidative folding and STT3-dependent N-glycosylation-so that only correctly folded proteins proceed through the secretory pathway. Impairment of protein load, folding capacity, or degradation via the ER-associated degradation (ERAD) pathway leads to the accumulation of unfolded proteins, triggering ER stress and activating the unfolded protein response (UPR), which, in the first instance, is an adaptive signaling network designed to restore homeostasis by adjusting protein synthesis, enhancing folding capacity, and promoting the clearance of misfolded proteins. During ER stress, the ER undergoes morphological and functional remodeling to manage the increased folding burden, including an increase of ER-mitochondria contact sites (ERMCs). These nanometric junctions (~10-100 nm) facilitate lipid and metabolite exchange and mediate calcium and reactive oxygen species signaling to support cellular metabolism. However, chronic ER stress can further tighten ERMCs, leading to calcium overload, mitochondrial dysfunction, and apoptosis. This review examines the core mechanisms underlying ER proteostasis in the context of ER stress and explores how ER stress first boosts mitochondrial activity and later impairs it through ERMCs, contributing to cell death and disease. Finally, emerging therapeutic strategies aimed at restoring proteostasis and modulating the dynamics of ERMCs are highlighted as promising interventions for conditions, such as cancer and congenital myopathies, where ER and mitochondrial dysfunction play central roles in pathogenesis.

Disulphide bonds (Dsbs) are essential for the folding, stability, and function of many secreted and membrane-associated proteins in bacteria. In Gram-negative species, these bonds are introduced by the Dsb enzyme family, with DsbA acting as the primary thiol oxidase. While DsbA proteins share a conserved thioredoxin (TRX)-like fold, emerging evidence highlights substantial structural and functional divergence among pathogenic homologues. Here, we present the high-resolution crystal structure and functional characterization of BperDsbA, a DsbA homologue from Bordetella pertussis, the causative agent of whooping cough. BperDsbA adopts a canonical TRX fold with a CPHC active site and a threonine-containing cis-proline loop, but displays striking deviations from prototypical DsbAs. Notably, it contains a highly destabilizing catalytic Dsb, resulting in one of the most oxidizing redox potentials recorded for a DsbA enzyme. Surface electrostatic analysis reveals an unusual distribution of positive and negative charge around the active site, in contrast to the broadly hydrophobic catalytic surfaces of other DsbAs. Functionally, BperDsbA shows limited substrate promiscuity and selectively catalyzes the oxidative folding of a pertussis toxin-derived peptide, supporting a model of substrate specialization. Together, these findings suggest that BperDsbA has evolved unique redox and structural features to support virulence factor maturation in B. pertussis. This work expands our understanding of the mechanistic diversity of DsbA enzymes and highlights their potential as pathogen-specific targets for anti-virulence therapeutics.

BACKGROUNDNeurodegeneration refers to the progressive loss of neurons, affecting both their structure and function. It is driven by synaptic dysfunction, disruptions in neural networks, and the accumulation of abnormal protein variants. Endoplasmic reticulum (ER) stress, caused by the accumulation of misfolded or unfolded protein, is a major contributor to neurodegeneration. Dithiothreitol (DTT) is a widely used redox reagent that disrupts the oxidative protein folding environment, inducing ER stress and leading to the imbalance in protein homeostasis can activate stress response pathway, potentially contributing to neurodegenerative processes. Caenorhabditis elegans (C. elegans) is a widely used model organism for studying neurodegeneration due to its well-mapped nervous system, approximately one-third of neuron cells in their body, complete genome sequenced, and conserved stress response pathway.AIMTo study the neurodegeneration in C. elegans caused by DTT-induced ER stress, assessed by behavioral, molecular, and lifespan changes.METHODSC. elegans were cultured on nematode growth medium plates with OP50, and ER stress was induced using DTT. Effects were assessed via behavioral assays such as locomotion, chemotaxis, lifespan assay, and molecular studies.RESULTSDTT exposure led to a significant decline in locomotion and chemotaxis response, indicating neurotoxicity. A reduction in lifespan was observed, suggesting an overall impact on health. Molecular analysis confirmed ER stress activation. DTT-induced ER stress negatively affects C. elegans, leading to behavioral impairments and molecular alterations associated with neurodegeneration.CONCLUSIONThese findings establish C. elegans as a potential model for studying ER stress-mediated neurotoxicity and its implications in neurodegenerative diseases.

In the last few decades, yeasts have been successfully engineered to be an excellent microbial cell factory for producing recombinant proteins with desired properties. This was due to their cost-effective characteristics and the successful application of genomic modification technologies. In addition, yeasts have a conserved post-translational modification pathway among eukaryotic organisms, which ensures the correct folding of recombinant proteins. However, the folding machinery cannot always cope with the load caused by the overexpression of recombinant genes, leading to the accumulation of misfolded proteins, the formation of aggregates and low production. Therefore, the protein-folding capacity of the endoplasmic reticulum (ER) remains one of the main limitations for heterologous protein production in yeast host organisms. However, thanks to many years of effective research of the fundamental mechanisms of protein folding, these limitations have been largely overcome. The study of folding in both model organisms and bioproducers has allowed to identify the molecular factors and cellular mechanisms that determine how a nascent polypeptide chain acquires its three-dimensional functional structure. This knowledge has become the basis for developing new effective techniques for engineering highly productive yeast strains. In this review, we examined the main cellular mechanisms associated with protein folding, such as ER transition, chaperone binding, oxidative folding, glycosylation, protein quality control. We discuss the effectiveness of applying this knowledge to the development of various engineering techniques aimed at overcoming bottlenecks in the protein folding system. In particular, selection of optimal signal peptides, co-expression with chaperones and foldases, modification of protein quality control, inhibition of proteolysis, and other techniques have allowed to enhance the ability of yeast bioproducers to effectively secrete heterologous proteins.

Pyrazolone-based ERO1 inhibitors in ERO1-driven triple-negative breast cancer and SEPN1-related myopathy: Structure-activity relationship and therapeutic potential.

Improving chemical synthesis and the antimicrobial activity of human defensins through disulfide bond engineering of HBD-3.

Cristae enclose respiratory chain complexes, making them the bioenergetic subcompartments of mitochondria. The Mitochondrial contact site and Cristae Organizing System (MICOS) complex is among the inducers of membrane curvature needed for crista formation. Resembling the respiratory chain complexes, MICOS is organized around a core protein, the mitofilin-domain bearing Mic60, that was inherited from the alphaproteobacterial progenitor of mitochondria. Extant alphaproteobacteria express Mic60 to form their own bioenergetic subcompartments, demonstrating the permeance of Mic60's form and function during prokaryotic and eukaryotic evolution. Yet, unlike virtually all aerobic eukaryotes, Mic60 is not encoded within the genomes of the multifarious protists that comprise the phylum Euglenozoa, including trypanosomes. Here, we show that Mic60 has been replaced in euglenozoans by two cryptic mitofilin domain-containing MICOS subunits, Mic34 and Mic40. Contrasting alphaproteobacterial and mitochondrial Mic60, these are not integral membrane proteins. Mic34 and Mic40 are as diverged from each other as both are to canonical Mic60. Reverse genetics revealed they are intertwined with the oxidative protein folding pathway required for mitochondrial–and crista–biogenesis, veiling a potential membrane remodeling role. Nevertheless, Mic34 binds phospholipid bilayers in vitro. Mic34 and Mic40 heterologous expression remodels gammaproteobacterial cytoplasmic membranes, like Mic60. Unexpectedly, Mic34 overexpression elaborates the simplified tubular mitochondrion of a Trypanosoma brucei life cycle stage with repressed oxidative phosphorylation. Furthermore, this activity was ablated by mutations to Mic34's mitofilin domain that correspond to essential motifs found in yeast Mic60's mitofilin domain. Thus, the mitofilin protein family is more diverse than originally supposed, with two of its structurally most divergent members altering the core of euglenozoan MICOS.

Characterization of the sTim/MIA pathway in Metamonada reveals different evolutionary adaptations to anaerobiosis.

The mitochondrial disulphide relay substrate FAM136A safeguards IMS proteostasis and cellular fitness

This study employed a comprehensive proteomic and metabolomic analysis to characterize adaptive cellular mechanisms of priority pathogens—Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, and Staphylococcus aureus—under sub-inhibitory concentrations of antibiotics. Despite significant metabolomic perturbations, some pathogens had minimal or no significant changes in their proteome. Notably, trimethylamine metabolism was consistently altered across all species, suggesting its role in survival under antibiotic stress. Shared adaptive responses to chloramphenicol in S. aureus and E. faecium are related to translation, oxidative stress management, protein folding and stability, biofilm formation capacity, glycine metabolism and osmoprotection. Alterations in quaternary amines and trimethylamine metabolism suggest alternative nitrogen and carbon utilization pathways in response to antibiotic stress. In S. aureus, vancomycin suppressed metabolism, including D-alanine metabolism, and global regulators LytR, CodY and CcpA. These findings offer insights into early antimicrobial resistance mechanisms and highlight critical proteins and metabolites linked to antibiotic tolerance.

Disulphide bond formation is critical for the folding and stability of proteins involved in bacterial cell envelope processes yet remains understudied in clostridial pathogens. While a few Clostridia-derived toxins and virulence factors are known to depend on disulphide bonds, the enzymes catalysing their formation are poorly characterized. Here, we performed a bioinformatic search to identify ten putative disulphide bond-forming enzymes in Clostridia. We cloned and codon-optimized these genes, testing their ability to complement Escherichia coli dsb mutants. Our analysis revealed a VKOR homologue, a VKOR-DsbA fusion and three DsbA homologues capable of complementing E. coli dsb mutants. Notably, Clostridium botulinum DsbA functioned independently of a regenerating partner, with its activity recycled by glutathione disulphide or ergothioneine. In contrast, Clostridium tetani and Clostridioides difficile DsbA proteins required E. coli DsbB for regeneration, suggesting reliance on distinct thiol or enzyme partners. Understanding oxidative protein folding in Clostridia could reveal new targets for antibacterial intervention.

Conotoxins are disulfide-rich peptides isolated from the venoms of marine cone snails. These natural products have inspired the development of several drug candidates and novel therapeutic leads. In addition to disulfide bonds, many conotoxins are highly modified with posttranslational modifications (PTMs) such as proline hydroxylation, C-terminal amidation and glycosylation, among others. These modifications can alter the charge, size and hydrophobicity of the conotoxin, influencing its interaction with target receptors and modulating its potency and selectivity. PTMs can also affect the folding kinetics and conformational stability of the peptide, which further affects its biological activity. Although conotoxins undergo a variety of PTMs, the functions of many of these modifications remain unclear. Here, we explored the structural and functional implications of PTMs in two representative conotoxins, PIIIA and TIIIA of the µ-pharmacological family. We synthesised a series of PIIIA and TIIIA peptides bearing native hydroxyproline and C-terminal amidation PTMs, along with their unmodified counterparts. Solid phase peptide synthesis and non-selective disulfide bond formation provided access to pure forms of the eight possible variants for in vitro comparison of their oxidative folding. Structural studies using nuclear magnetic resonance (NMR) spectroscopy, alongside electrophysiological and serum stability assays, were conducted to characterise the functional roles of the PTMs in these conotoxins. Our results suggest that, whereas C-terminal amidation has a crucial role in folding and structural integrity, proline hydroxylation significantly influences the in vitro oxidative folding, stability and biological activity of these conotoxin peptides.

Mycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-possess a unique and impermeable cell envelope that is critical for survival and antibiotic resistance. The assembly and maintenance of this envelope depend on properly folded proteins, yet the role of disulfide bond formation in these processes remains poorly understood. Mycobacteria rely on two membrane enzymes, disulfide bond formation protein A (DsbA) and vitamin K epoxide reductase (VKOR), for introducing disulfide bonds into exported proteins. In silico studies predict that ~64% of exported proteins contain even numbers of cysteine residues and thence disulfide bonding; nevertheless, substrates of the DsbA-VKOR pathway remain largely unknown. Here, we demonstrate that DsbA and VKOR introduce disulfide bonds into substrate proteins and identify several essential proteins that depend on oxidative folding in the mycobacterial cell envelope. Using bioinformatics and cysteine profiling proteomics, we uncover numerous exported proteins that require disulfide bonds for stability. Cysteine derivatization in whole cells confirms that key proteins, including LamA (MmpS3), PstP, LpqW, and EmbB, rely on disulfide bonds for proper function. Furthermore, chemical inhibition of VKOR phenocopies vkor deletion, thus highlighting its essential role in maintaining mycomembrane integrity. These findings address a critical gap in understanding mycobacterial cell envelope biogenesis and underscore the DsbA-VKOR system as a promising target for disrupting cell envelope homeostasis in drug-resistant Mycobacteria.IMPORTANCEThis work addresses a major deficiency in understanding mycobacterial cell envelope processes and highlights the biological and clinical implications of oxidative protein folding in mycobacteria. This process, marked by the formation of disulfide bonds, is essential for the stability of exported proteins. While disulfide bond formation studies in Gram-negative bacteria suggested a similar role in mycobacteria, the underlying consequences of disulfide bonds remained unclear. Thus, we began investigating the diverse physiological functions dependent on disulfide bonds in Mycobacteria using a combination of bioinformatics, proteomics, and genetic and biochemical approaches. We identified hundreds of proteins affected by oxidative protein folding and validated essential substrates of this process. We show that disulfide bonds are not only crucial for the stability and function of key mycobacterial proteins but also represent a novel therapeutic target against antimicrobial resistance. Our findings underscore the potential of targeting disulfide bond formation to disrupt mycomembrane assembly, opening new avenues for antimycobacterial drug development.

BackgroundM2 macrophages are known to be involved in tumorigenesis. However, the mechanism by which they promote tumor progression in endometrial cancer (EC) remains largely unknown. Kynureninase (KYNU) has been found to be associated with the progression of various tumors, but research on endometrium is limited to embryo transfer. Therefore, a better understanding of KYNU as a potential therapeutic target in EC treatment is needed. This study aimed to elucidate the mechanism by which M2 macrophage-secreted KYNU influences the malignant biological and stemness remodeling of EC via the SOD2-mtROS-ERO1α and endoplasmic reticulum unfolded protein response (UPRER) pathway.MethodsWe used flow cytometry for cell sorting. Fluorescence experiments were conducted to reveal spatial position of protein, and. Western blot and qRT‒PCR were used to detect the protein and mRNA levels, respectively. The interaction between KYNU and superoxide dismutase 2 (SOD2) was demonstrated using coimmunoprecipitation experiments. Furthermore, the mechanism between activating transcription factor 4 (ATF4) and the KYNU was assessed using chromatin immunoprecipitation and dual luciferase assays. Cell Counting Kit-8, flow cytometry, and transwell assays were used to detect tumor cell proliferation, apoptosis, and invasion capacities. Student’s t test and one-way analysis of variance (ANOVA) were used to compare groups.ResultsM2 macrophage-secreted KYNU induced malignant behavior and stemness via the SOD2-mtROS-ERO1α-UPRER pathway, contributing to a positive feedback loop for tumor cell self-protection. Mechanistically, KYNU and its metabolite 3-hydroxyanthranillic acid (3-HAA) upregulated the expression of SOD2, thereby decreasing mitochondrial reactive oxygen species (mtROS). KYNU inhibitors affected the spatial overflow of mtROS from mitochondria to the endoplasmic reticulum (ER). Endoplasmic reticulum oxidoreductin 1α (ERO1α) was sensitively affected by KYNU-induced changes in the redox environment, stimulating the PERK-eIF2α-ATF4 pathway of the UPRER. This in turn promoted oxidative folding, reduced the level of misfolded protein (MFP), and maintained tumor survival and progression. Additionally, ATF4 acted as a transcription factor in the KYNU promoter region, amplifying KYNU tumorigenesis in a positive feedback manner.ConclusionM2-secreted KYNU promotes the malignant behavior and stemness remodeling of EC via the SOD2-mtROS-ERO1α-UPRER axis and establishes a positive feedback loop. Thus, KYNU is a potential therapeutic target for EC treatment.

Disulfide-rich peptides (DRPs), particularly those featuring the inhibitor cystine knot (ICK) motif, represent promising scaffolds for developing next-generation protein modulators and therapeutic agents due to their remarkable stability and specificity. However, their inherent structural integrity and lack of structural plasticity significantly limit their evolvability, creating a fundamental bottleneck in engineering novel functionalities. To address this challenge, we developed a novel proline scanning strategy aimed at enhancing the evolvability of the ICK scaffolds. This strategy leverages the proline-mediated structural decoupling between scaffold and nonscaffold residues in DRPs to promote their evolvability. By strategically incorporating prolines as pre-encoded scaffold residues, we engineered ICK variants with significantly improved foldability and tolerance to sequence variations. This advancement enabled the construction of diverse peptide libraries suitable for screening platforms, including mRNA and phage display. Utilizing this approach, we successfully identified DRPs exhibiting low-nanomolar affinity to clinically important targets, such as TROP2 and 4-1BB. Structural characterization revealed that these evolved DRPs adopted unique three-dimensional structures stabilized by up to four disulfide bonds, demonstrating both high oxidative folding efficiency and enhanced evolvability due to proline incorporation. To evaluate their therapeutic potential, we developed a DRP-based chimeric antigen receptor (CAR) targeting TROP2. The DRP-based CAR T cells exhibited potency comparable to conventional single-chain variable fragment (scFv)-based CAR T cells but with a notably improved safety profile. Overall, our work establishes a robust framework for expanding the functional versatility of DRP scaffolds, facilitating the discovery and development of structurally diverse and functional DRPs for broad applications in therapeutics and drug development.

Cover Feature: Oxidative Folding of a Two‐Chain Protein Having Three Interchain Disulfide Bonds. Synthesis of Bromelain Inhibitor VI Through Native Chain Assembly

Dissecting oxidative folding of conotoxins using 3D structures of cysteine mutants predicted by AlphaFold 3: A case study of α-conotoxin RgIA, χ-conotoxin CMrVIA and ω-conotoxin MVIIA-Gly.

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 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.

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.