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The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of soluble proteins into the compartment, where they are retained through oxidative folding. Additionally, the disulfide relay enhances the stability of many proteins by forming disulfide bonds. In this review, we describe the mitochondrial disulfide relay in human cells, its components, and their coordinated collaboration in mechanistic detail. We also discuss the human pathologies associated with defects in this machinery and its protein substrates, providing a comprehensive overview of its biological importance and implications for health. [ABSTRACT FROM AUTHOR]

The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of soluble proteins into the compartment, where they are retained through oxidative folding. Additionally, the disulfide relay enhances the stability of many proteins by forming disulfide bonds. In this review, we describe the mitochondrial disulfide relay in human cells, its components, and their coordinated collaboration in mechanistic detail. We also discuss the human pathologies associated with defects in this machinery and its protein substrates, providing a comprehensive overview of its biological importance and implications for health.

Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a′, at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H2O2-sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a′ active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a′ active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown. [ABSTRACT FROM AUTHOR]

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by‐product is hydrogen peroxide (H2O2). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER‐derived H2O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging‐related disease intervention. Synopsis: Protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulates in aged human mesenchymal stem cells. Depletion of PDI slows the rate of oxidative protein folding, decreases the leakage of ER‐derived H2O2 into the nucleus, and alleviates cell senescence. PDI accumulates in aged human mesenchymal stem cells and depletion of PDI alleviates cell senescence.PDI deficiency slows the rate of oxidative protein folding and reduces the level of its byproduct H2O2 in both the ER and nucleus.Increased H2O2 levels induce the expression of SERPINE1, a component of the senescence‐associated secretory phenotype (SASP), which accelerates cell senescence. [ABSTRACT FROM AUTHOR]

Simple Summary: The attachment of sugar residues to proteins is known as glycosylation. Glycosylation is a stepwise process which is significantly altered during cancer and may thus serve as both a therapeutic and/or diagnostic target. However, our understanding of how and which glycan structures contribute to aggressive cancer behaviors is lacking. In this study, we examined how specific N-glycans contribute to aggressive neuroblastoma behavior. Here, we use genetic editing to direct N-glycan expression in neuroblastoma cells, thus enabling us to assign specific N-glycan structures to cellular properties of neuroblastoma. In addition, we also examine how altering the process of N-glycosylation influences the expression of endoplasmic reticulum (ER) folding and transport proteins. It is critical to understand how N-glycosylation processing may influence ER folding and transport protein expression. This study demonstrates that alterations of N-glycosylation influence neuroblastoma progression by diminishing or enhancing cellular properties associated with cancer progression, along with influencing the expression pattern of ER folding and transport proteins. Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6–9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, oxidative protein folding occurs in the lumen of the endoplasmic reticulum (ER), catalyzed by ER sulfhydryl oxidase 1 (Ero1) and protein disulfide isomerase (PDI). The efficiency and fidelity of oxidative protein folding are vital for the function of secretory cells. Here, we summarize oxidative protein folding in yeast, plants, and mammals, and discuss how the conformation and activity of human Ero1-PDI machinery is regulated through various post-translational modifications (PTMs). We propose that oxidative protein folding fidelity and ER redox homeostasis are maintained by both the precise control of Ero1 oxidase activity and the division of labor between PDI family members. We also discuss how deregulated Ero1-PDI functions contribute to human diseases and can be leveraged for therapeutic interventions. The endoplasmic reticulum (ER) sulfhydryl oxidase 1 (Ero1) and protein disulfide isomerase (PDI) family constitute a conserved and pivotal pathway through which oxidative protein folding is catalyzed in the ER of eukaryotic cells. Loss of Ero1 activity in different species causes intolerance to reductive stress, while loss of a PDI protein usually results in the failure of native disulfide formation. The conformation and activity of the human Ero1-PDI machinery is regulated by various PTMs, and therefore this machine sensitively responds to redox fluctuations in the ER. Different proteins of the PDI family exhibit a different division of labor during oxidative protein folding and work synergistically to guarantee efficient and faithful disulfide formation. Deregulated Ero1-PDI functions contribute to various human diseases, including diabetes, cancers, and cardiovascular diseases, and therefore Ero1-PD1 may be a target for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4 −/− mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4 −/− mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis. • Granulosa cell endoplasmic reticulum proteostasis is pivotal for ovarian aging. • PRDX4 protects against ovarian aging by sustaining granulosa cell ER proteostasis. • Deficiency of PRDX4 has a detrimental impact on the expression of FSHR. [ABSTRACT FROM AUTHOR]

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5-disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.

Peroxiredoxin IV (Prx4) is a 2-Cysteine peroxidase with ubiquitous expression in human tissues. Prx4 scavenges hydrogen peroxide and participates in oxidative protein folding in the endoplasmic reticulum. In addition, Prx4 is secreted outside the cell. Prx4 is upregulated in several cancers and is a potential therapeutic target. We have summarized historical and recent advances in the structure, function and biological roles of Prx4, focusing on inflammatory diseases and cancer. Oxidative stress is known to activate pro-inflammatory pathways. Chronic inflammation is a risk factor for cancer development. Hence, redox enzymes such as Prx4 are important players in the crosstalk between inflammation and cancer. Understanding molecular mechanisms of regulation of Prx4 expression and associated signaling pathways in normal physiological and disease conditions should reveal new therapeutic strategies. Thus, although Prx4 is a promising therapeutic target for inflammatory diseases and cancer, further research needs to be conducted to bridge the gap to clinical application. [ABSTRACT FROM AUTHOR]

Peroxiredoxin IV (Prx4) is a 2-Cysteine peroxidase with ubiquitous expression in human tissues. Prx4 scavenges hydrogen peroxide and participates in oxidative protein folding in the endoplasmic reticulum. In addition, Prx4 is secreted outside the cell. Prx4 is upregulated in several cancers and is a potential therapeutic target. We have summarized historical and recent advances in the structure, function and biological roles of Prx4, focusing on inflammatory diseases and cancer. Oxidative stress is known to activate pro-inflammatory pathways. Chronic inflammation is a risk factor for cancer development. Hence, redox enzymes such as Prx4 are important players in the crosstalk between inflammation and cancer. Understanding molecular mechanisms of regulation of Prx4 expression and associated signaling pathways in normal physiological and disease conditions should reveal new therapeutic strategies. Thus, although Prx4 is a promising therapeutic target for inflammatory diseases and cancer, further research needs to be conducted to bridge the gap to clinical application.

Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine‐containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision‐making workflow for exploiting disulfide bond formation for the expression of any given protein of interest. [ABSTRACT FROM AUTHOR]

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

The folding of proteins that contain disulfide bonds is termed oxidative protein folding. It involves a chemical reaction resulting in the formation of disulfide bonds and a physical conformational folding reaction that promotes the formation of the native structure. While the presence of disulfide bonds significantly increases the complexity of the folding landscape, it is generally recognized that native disulfide bonds help funnel the trajectory towards the final folded form. Here, we review the role of disulfide bonds in oxidative protein folding and argue that even structure-inducing native disulfide bond formation treads a fine line in the regeneration of disulfide-bond-containing proteins. The translation of this observation to protein misfolding related disorders is discussed. [ABSTRACT FROM AUTHOR]

Maintenance of cellular redox control is pivotal for normal cellular functions and cell fate decisions including cell death. Among the key cellular redox systems in mammals, the glutathione peroxidase (GPX) family of proteins is the largest conferring multifaceted functions and affecting virtually all cellular processes. The endoplasmic reticulum (ER)-resident GPXs, designated as GPX7 and GPX8, are the most recently added members of this family of enzymes. Recent studies have provided exciting insights how both enzymes support critical processes of the ER including oxidative protein folding, maintenance of ER redox control by eliminating H2O2, and preventing palmitic acid-induced lipotoxicity. Consequently, numerous pathological conditions, such as neurodegeneration, cancer and metabolic diseases have been linked with altered GPX7 and GPX8 expression. Studies in mice have demonstrated that loss of GPX7 leads to increased differentiation of preadipocytes, increased tumorigenesis and shortened lifespan. By contrast, GPX8 deficiency in mice results in enhanced caspase-4/11 activation and increased endotoxic shock in colitis model. With the increasing recognition that both types of enzymes are dysregulated in various tumor entities in man, we deem a review of the emerging roles played by GPX7 and GPX8 in health and disease development timely and appropriate. [ABSTRACT FROM AUTHOR]

The formation and isomerization of disulfide bonds mediated by protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) is of fundamental importance in eukaryotes. Canonical PDI structure comprises four domains with the order of a-b-b′-a′. In Arabidopsis thaliana, the PDI-S subgroup contains only one member, AtPDI11, with an a-a′-D organization, which has no orthologs in mammals or yeast. However, the expression pattern of AtPDI11 and the functioning mechanism of AtPDI11 D domain are currently unclear. In this work, we found that PDI-S is evolutionarily conserved between land plants and algal organisms. AtPDI11 is expressed in various tissues and its induction by ER stress is disrupted in bzip28/60 and ire1a/b mutants that are null mutants of key components in the unfolded protein response (UPR) signal transduction pathway, suggesting that the induction of AtPDI11 by ER stress is mediated by the UPR signaling pathway. Furthermore, enzymatic activity assays and genetic evidence showed that the D domain is crucially important for the activities of AtPDI11. Overall, this work will help to further understand the working mechanism of AtPDI11 in catalyzing disulfide formation in plants.

Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErvC17S. Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant LtErv constructs were able to rescue yeast cells that lack ScMia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum. Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies.

Long‐acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se‐insulin[C7UA, C7UB] was reported to be protected from proteolysis by insulin‐degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu‐C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid‐acting clinical analogue (insulin lispro). Although [C7UA, C7UB] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight. [ABSTRACT FROM AUTHOR]

Abstract Background Many proteins of the mitochondrial intermembrane space (IMS) contain structural disulfide bonds formed by the mitochondrial disulfide relay. In fungi and animals, the sulfhydryl oxidase Erv1 ‘generates’ disulfide bonds that are passed on to the oxidoreductase Mia40, which oxidizes substrate proteins. A different structural organization of plant Erv1 proteins compared to that of animal and fungal orthologs was proposed to explain its inability to complement the corresponding yeast mutant. Results Herein, we have revisited the biochemical and functional properties of Arabidopsis thaliana Erv1 by both in vitro reconstituted activity assays and complementation of erv1 and mia40 yeast mutants. These mutants were viable, however, they showed severe defects in the biogenesis of IMS proteins. The plant Erv1 was unable to oxidize yeast Mia40 and rather even blocked its activity. Nevertheless, it was able to mediate the import and folding of mitochondrial proteins. Conclusions We observed that plant Erv1, unlike its homologs in fungi and animals, can promote protein import and oxidative protein folding in the IMS independently of the oxidoreductase Mia40. In accordance to the absence of Mia40 in many protists, our study suggests that the mitochondrial disulfide relay evolved in a stepwise reaction from an Erv1-only system to which Mia40 was added in order to improve substrate specificity. Graphical Abstract The mitochondrial disulfide relay evolved in a step-wise manner from an Erv1-only system.

Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H2O2 sensor in yeast, is present in the mitochondrial intermembrane space (IMS), where it serves a compartment-specific role in oxidative metabolism. The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis.

Oxidation of cysteine residues in proteins can take place as part of an enzymatic reaction cycle, during oxidative protein folding or as a consequence of redox signalling or oxidative stress. Following changes in protein thiol redox states allows to investigate the mechanisms underlying thiol-disulphide redox processes. In this book chapter, we provide information and protocols on different methods for redox state determination with a focus on these processes in the context of oxidation-dependent protein import into the mitochondrial intermembrane space. These methods include assessing the cysteine redox state of mature proteins, methods to investigate oxidative protein folding in radioactive pulse chase assays and methods to follow specifically the formation of oxidative folding intermediates between oxidoreductases and substrates., (Copyright © 2024. Published by Elsevier Inc.)

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6 and CysA11 (which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid‐phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild‐type B chain‐di‐S‐sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long‐standing challenge in chemical insulin synthesis. The affinity of the Se‐insulin analogue for the lectin‐purified insulin receptor was indistinguishable from that of WT‐insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu ≈0.8 kcal mol−1). In accordance with such enhanced stability, reductive unfolding of the Se‐insulin analogue and resistance to enzymatic cleavage by Glu‐C protease occurred four times more slowly than that of WT‐insulin. 2D‐NMR and X‐ray crystallographic studies demonstrated a native‐like three‐dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash. [ABSTRACT FROM AUTHOR]

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described. Oxidative protein folding in the plant endoplasmic reticulum [ABSTRACT FROM AUTHOR]

The mitochondrial intermembrane space evolved from the bacterial periplasm. Presumably as a consequence of their common origin, most proteins of these compartments are stabilized by structural disulfide bonds. The molecular machineries that mediate oxidative protein folding in bacteria and mitochondria, however, appear to share no common ancestry. Here we tested whether the enzymes Erv1 and Mia40 of the yeast mitochondrial disulfide relay could be functionally replaced by corresponding components of other compartments. We found that the sulfhydryl oxidase Erv1 could be replaced by the Ero1 oxidase or the protein disulfide isomerase from the endoplasmic reticulum, however at the cost of respiration deficiency. In contrast to Erv1, the mitochondrial oxidoreductase Mia40 proved to be indispensable and could not be replaced by thioredoxin-like enzymes, including the cytoplasmic reductase thioredoxin, the periplasmic dithiol oxidase DsbA, and Pdi1. From our studies we conclude that the profound inertness against glutathione, its slow oxidation kinetics and its high affinity to substrates renders Mia40 a unique and essential component of mitochondrial biogenesis. Evidently, the development of a specific mitochondrial disulfide relay system represented a crucial step in the evolution of the eukaryotic cell. [ABSTRACT FROM AUTHOR]

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol‐disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active‐site motif embedded in their thioredoxin‐like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol‐disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram‐negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms. [ABSTRACT FROM AUTHOR]

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.

Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

In most cellular compartments cysteine residues are predominantly reduced. However, in the bacterial periplasm, the ER and the mitochondrial intermembrane space (IMS), sulfhydryl oxidases catalyze the formation of disulfide bonds. Nevertheless, many IMS proteins contain reduced cysteines that participate in binding metal- or heme-cofactors. In this study, we addressed the substrate specificity of the mitochondrial protein oxidation machinery. Dre2 is a cysteine-rich protein that is located in the cytosol. A large fraction of Dre2 bound to the cytosolic side of the outer membrane of mitochondria. Even when Dre2 is artificially targeted to the IMS, its cysteine residues remain in the reduced state. This indicates that protein oxidation in the IMS of mitochondria is not a consequence of the apparent oxidizing environment in this compartment but rather is substrate-specific and determined by the presence of Mia40-binding sites.

Abstract: Family with sequence similarity 20C (Fam20C), the physiological Golgi casein kinase, phosphorylates numerous secreted proteins that are involved in a wide variety of biological processes. However, the role of Fam20C in regulating proteins in the endoplasmic reticulum (ER) lumen is largely unknown. Here, we report that Fam20C interacts with various luminal proteins and that its depletion results in a more reduced ER lumen. We further show that ER oxidoreductin 1α (Ero1α), the pivotal sulfhydryl oxidase that catalyzes disulfide formation in the ER, is phosphorylated by Fam20C in the Golgi apparatus and retrograde‐transported to the ER mediated by ERp44. The phosphorylation of Ser145 greatly enhances Ero1α oxidase activity and is critical for maintaining ER redox homeostasis and promoting oxidative protein folding. Notably, phosphorylation of Ero1α is induced under hypoxia, reductive stress, and secretion‐demanding conditions such as mammalian lactation. Collectively, our findings open a door to uncover how oxidative protein folding is regulated by phosphorylation in the secretory pathway. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are produced in various cell compartments by an array of enzymes and processes. An excess of ROS production can be hazardous for normal cell functioning, whereas at normal levels, ROS act as vital regulators of many signal transduction pathways and transcription factors. ROS production is affected by a wide range of viruses. However, to date, the impact of viral infections has been studied only in respect to selected ROS-generating enzymes. The role of several ROS-generating and -scavenging enzymes or cellular systems in viral infections has never been addressed. In this review, we focus on the roles of biogenic polyamines and oxidative protein folding in the endoplasmic reticulum (ER) and their interplay with viruses. Polyamines act as ROS scavengers, however, their catabolismis accompanied by H2O2 production. Hydrogen peroxide is also produced during oxidative protein folding, with ER oxidoreductin 1 (Ero1) being a major source of oxidative equivalents. In addition, Ero1 controls Ca2+ efflux from the ER in response to e.g., ER stress. Here, we briefly summarize the current knowledge on the physiological roles of biogenic polyamines and the role of Ero1 at the ER, and present available data on their interplay with viral infections. [ABSTRACT FROM AUTHOR]

Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Background: Many proteins of the mitochondrial intermembrane space (IMS) contain structural disulfide bonds formed by the mitochondrial disulfide relay. In fungi and animals, the sulfhydryl oxidase Erv1 'generates' disulfide bonds that are passed on to the oxidoreductase Mia40, which oxidizes substrate proteins. A different structural organization of plant Erv1 proteins compared to that of animal and fungal orthologs was proposed to explain its inability to complement the corresponding yeast mutant. Results: Herein, we have revisited the biochemical and functional properties of Arabidopsis thaliana Erv1 by both in vitro reconstituted activity assays and complementation of erv1 and mia40 yeast mutants. These mutants were viable, however, they showed severe defects in the biogenesis of IMS proteins. The plant Erv1 was unable to oxidize yeast Mia40 and rather even blocked its activity. Nevertheless, it was able to mediate the import and folding of mitochondrial proteins. Conclusions: We observed that plant Erv1, unlike its homologs in fungi and animals, can promote protein import and oxidative protein folding in the IMS independently of the oxidoreductase Mia40. In accordance to the absence of Mia40 in many protists, our study suggests that the mitochondrial disulfide relay evolved in a stepwise reaction from an Erv1-only system to which Mia40 was added in order to improve substrate specificity. [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI. [ABSTRACT FROM AUTHOR]

Summary Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H 2 O 2 sensor in yeast, is present in the mitochondrial intermembrane space (IMS), where it serves a compartment-specific role in oxidative metabolism. The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis. [ABSTRACT FROM AUTHOR]

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H 2 O 2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H 2 O 2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention., (© 2023 The Authors.)

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.

It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64–80] and des[6–127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5–45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1–4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1–4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76–94] was populated in the largest amount, but the oxidation to N was slower than that of des[64–80] and des[6–127]. At 35 °C, on the other hand, des[64–80] and des[6–127] were no longer stable, and only des[76–94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control.

Oxidative folding in the mitochondrial intermembrane space (IMS) is a key cellular event associated with the folding and import of a large and still undetermined number of proteins. This process is catalyzed by an oxidoreductase, Mia40 that is able to recognize substrates with apparently little or no homology. Following substrate oxidation, Mia40 is reduced and must be reoxidized by Erv1/Alr1 that consequently transfers the electrons to the mitochondrial respiratory chain. Although our understanding of the physiological relevance of this process is still limited, an increasing number of pathologies are being associated with the impairment of this pathway; especially because oxidative folding is fundamental for several of the proteins involved in defense against oxidative stress. Here we review these aspects and discuss recent findings suggesting that oxidative folding in the IMS is modulated by the redox state of the cell.

Reactive oxygen species (ROS) are produced in various cell compartments by an array of enzymes and processes. An excess of ROS production can be hazardous for normal cell functioning, whereas at normal levels, ROS act as vital regulators of many signal transduction pathways and transcription factors. ROS production is affected by a wide range of viruses. However, to date, the impact of viral infections has been studied only in respect to selected ROS-generating enzymes. The role of several ROS-generating and -scavenging enzymes or cellular systems in viral infections has never been addressed. In this review, we focus on the roles of biogenic polyamines and oxidative protein folding in the endoplasmic reticulum (ER) and their interplay with viruses. Polyamines act as ROS scavengers, however, their catabolism is accompanied by H2O2 production. Hydrogen peroxide is also produced during oxidative protein folding, with ER oxidoreductin 1 (Ero1) being a major source of oxidative equivalents. In addition, Ero1 controls Ca2+ efflux from the ER in response to e.g., ER stress. Here, we briefly summarize the current knowledge on the physiological roles of biogenic polyamines and the role of Ero1 at the ER, and present available data on their interplay with viral infections.

Mitochondria are fundamental organelles with a complex internal architecture that fulfill important diverse functions including iron–sulfur cluster assembly and cell respiration. Intense work for more than 30 years has identified the key protein import components and the pathways involved in protein targeting and assembly. More recently, oxidative folding has been discovered as one important mechanism for mitochondrial proteostasis whilst several human disorders have been linked to this pathway. We describe the molecular components of this pathway in view of their putative redox regulation and we summarize available evidence on the connections of these pathways to human disorders. [ABSTRACT FROM AUTHOR]

Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40’s ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (PRDXs) form an enzyme family that exhibits peroxidase activity using electrons from thioredoxin and other donor molecules. As the signaling roles of hydrogen peroxide in response to extracellular stimuli have emerged, the involvement of PRDX in the hydrogen peroxide-mediated signaling has become evident. Among six PRDX members in mammalian cells, PRDX4 uniquely possesses a hydrophobic signal peptide at the amino terminus, and, hence, it undergoes either secretion or retention by the endoplasmic reticulum (ER) lumen. The role of PRDX4 as a sulfoxidase in ER is now attracting much attention regarding the oxidative protein folding of nascent proteins. Contrary to this role in the ER, the functional significance of PRDX4 in the extracellular milieu is virtually unknown despite its implications as a biomarker under pathological conditions in some diseases. Other than its systemically expressed form, a variant form of PRDX4 is transcribed from the upstream promoter/exon 1 of the systemic promoter/exon 1 and is uniquely expressed in sexually matured testes. Circumstantial evidence, together with deduced functions from the systemic form, suggests that there are potential roles for testicular PRDX4 in the reproductive processes such as the regulation of hormonal signals and the oxidative packaging of sperm chromatin. Elucidation of these PRDX4 functions under in vivo situations is expected to show the whole picture of how PRDX4 has evolved in multicellular organisms. [ABSTRACT FROM AUTHOR]

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

Protein disulfide–isomerase (PDI) was the first protein-folding catalyst to be characterized, half a century ago. It plays critical roles in a variety of physiological events by displaying oxidoreductase and redox-regulated chaperone activities. This review provides a brief history of the identification of PDI as both an enzyme and a molecular chaperone and of the recent advances in studies on the structure and dynamics of PDI, the substrate binding and release, and the cooperation with its partners to catalyze oxidative protein folding and maintain ER redox homeostasis. In this review, we highlight the structural features of PDI, including the high interdomain flexibility, the multiple binding sites, the two synergic active sites, and the redox-dependent conformational changes. [ABSTRACT FROM AUTHOR]