Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway

Disulfide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulfide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulfide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulfide bond formation is reviewed with emphasis on events that occur during ER entry. Disulfide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulfide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulfide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.

Download Full-text

Two phases of disulfide bond formation have differing requirements for oxygen

The Journal of Cell Biology ◽

10.1083/jcb.201307185 ◽

2013 ◽

Vol 203 (4) ◽

pp. 615-627 ◽

Cited By ~ 64

Author(s):

Marianne Koritzinsky ◽

Fiana Levitin ◽

Twan van den Beucken ◽

Ryan A. Rumantir ◽

Nicholas J. Harding ◽

...

Keyword(s):

Er Stress ◽

Disulfide Bond ◽

Electron Acceptor ◽

Disulfide Bonds ◽

Mammalian Cells ◽

Secretory Pathway ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Terminal Electron Acceptor ◽

Two Phases

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.

Download Full-text

In situ detection of protein interactions for recombinant therapeutic enzymes

10.1101/2020.05.06.081885 ◽

2020 ◽

Author(s):

Mojtaba Samoudi ◽

Chih-Chung Kuo ◽

Caressa M. Robinson ◽

Km Shams-Ud-Doha ◽

Song-Min Schinn ◽

...

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Ppi Networks

AbstractDespite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.

Download Full-text

Ero1–PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0403 ◽

2013 ◽

Vol 368 (1617) ◽

pp. 20110403 ◽

Cited By ~ 45

Author(s):

Adam M. Benham ◽

Marcel van Lith ◽

Roberto Sitia ◽

Ineke Braakman

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Secretory Pathway ◽

Protein Complexes ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Terminal Electron Acceptor ◽

Post Translational Modifications

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide bonds. These are introduced into client proteins by ER resident oxidoreductases, including ER oxidoreductin 1 (Ero1). Ero1 is usually considered to function in a linear pathway, by ‘donating’ a disulfide bond to protein disulfide isomerase (PDI) and receiving electrons that are passed on to the terminal electron acceptor molecular oxygen. PDI engages with a range of clients as the direct catalyst of disulfide bond formation, isomerization or reduction. In this paper, we will consider the interactions of Ero1 with PDI family proteins and chaperones, highlighting the effect that redox flux has on Ero1 partnerships. In addition, we will discuss whether higher order protein complexes play a role in Ero1 function.

Download Full-text

Functional studies of per os infectivity factor 3 of Helicoverpa armigera nucleopolyhedrovirus

Journal of General Virology ◽

10.1099/vir.0.035865-0 ◽

2012 ◽

Vol 93 (2) ◽

pp. 374-382 ◽

Cited By ~ 5

Author(s):

Jingjiao Song ◽

Manli Wang ◽

Huachao Huang ◽

Xin Luo ◽

Fei Deng ◽

...

Keyword(s):

Disulfide Bond ◽

Helicoverpa Armigera ◽

Disulfide Bonds ◽

Bond Formation ◽

Terminal Region ◽

Disulfide Bond Formation ◽

Functional Studies ◽

Nuclear Ring ◽

Helicoverpa Armígera ◽

And Function

PIF3 is one of the six conserved per os infectivity factors (PIFs) of baculoviruses. In this study, PIF3 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was analysed by infectivity bioassays using a series of recombinant viruses harbouring various PIF3 truncation/substitution mutants. The results demonstrated that the N-terminal region (L26–Y45) and C-terminal region (T160–Q199) are essential for HearNPV oral infectivity. In the C-terminal T160–Q199 region, there are three conserved cysteines (C162, C164 and C185). Our results showed that substitutions of C162 or C164, predicted to be involved in disulfide-bond formation, led to a severe decrease in HearNPV per os infectivity. Mutation of C185, predicted not to be involved in disulfide-bond formation, did not affect the per os infectivity. The data suggest that disulfide bonds are important for PIF3 conformation and function. Immunofluorescence assays showed that none of the mutations affected the subcellular localization of PIF3 to the nuclear ring zone region of infected cells. Western blot results showed that all mutants except C162G and C185G failed to incorporate PIF3 into occlusion-derived viruses, which resulted in impaired oral infectivity of the latter. The data provide insights for future study of PIF3 function.

Download Full-text

Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

Journal of Virology ◽

10.1128/jvi.02295-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6464-6476 ◽

Cited By ~ 18

Author(s):

Yao-Cheng Ching ◽

Che-Sheng Chung ◽

Cheng-Yen Huang ◽

Yu Hsia ◽

Yin-Liang Tang ◽

...

Keyword(s):

Vaccinia Virus ◽

Cell Fusion ◽

Disulfide Bond ◽

Protein Complex ◽

Disulfide Bonds ◽

Bond Formation ◽

In Vitro Mutagenesis ◽

Disulfide Bond Formation ◽

Infected Cells

ABSTRACT Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

Download Full-text

A Secreted Disulfide Catalyst Controls Extracellular Matrix Composition and Function

Science ◽

10.1126/science.1238279 ◽

2013 ◽

Vol 341 (6141) ◽

pp. 74-76 ◽

Cited By ~ 83

Author(s):

Tal Ilani ◽

Assaf Alon ◽

Iris Grossman ◽

Ben Horowitz ◽

Elena Kartvelishvily ◽

...

Keyword(s):

Extracellular Matrix ◽

Disulfide Bond ◽

De Novo ◽

Bond Formation ◽

Supporting Cell ◽

Matrix Composition ◽

Secretory Proteins ◽

Disulfide Bond Formation ◽

Enzyme Families ◽

And Function

Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.

Download Full-text

Disulfide formation in plant storage vacuoles permits assembly of a multimeric lectin

Biochemical Journal ◽

10.1042/bj20091878 ◽

2010 ◽

Vol 427 (3) ◽

pp. 513-521 ◽

Cited By ~ 7

Author(s):

Richard S. Marshall ◽

Lorenzo Frigerio ◽

Lynne M. Roberts

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Castor Oil ◽

Ricinus Communis ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Endosperm Tissue ◽

Oil Seed ◽

Protein Storage Vacuole ◽

Protein Disulfide

The ER (endoplasmic reticulum) has long been considered the plant cell compartment within which protein disulfide bond formation occurs. Members of the ER-located PDI (protein disulfide isomerase) family are responsible for oxidizing, reducing and isomerizing disulfide bonds, as well as functioning as chaperones to newly synthesized proteins. In the present study we demonstrate that an abundant 7S lectin of the castor oil seed protein storage vacuole, RCA (Ricinus communis agglutinin 1), is folded in the ER as disulfide bonded A–B dimers in both vegetative cells of tobacco leaf and in castor oil seed endosperm, but that these assemble into (A–B)2 disulfide-bonded tetramers only after Golgi-mediated delivery to the storage vacuoles in the producing endosperm tissue. These observations reveal an alternative and novel site conducive for disulfide bond formation in plant cells.

Download Full-text

Cardiac peroxiredoxins undergo complex modifications during cardiac oxidant stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00017.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. H425-H433 ◽

Cited By ~ 37

Author(s):

Ewald Schröder ◽

Jonathan P. Brennan ◽

Philip Eaton

Keyword(s):

Hydrogen Peroxide ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Structural Changes ◽

Oxidant Stress ◽

Bond Formation ◽

Redox Signaling ◽

Size Exclusion ◽

Disulfide Bond Formation ◽

Dimeric Unit

Peroxiredoxins (Prdxs), a family of antioxidant and redox-signaling proteins, are plentiful within the heart; however, their cardiac functions are poorly understood. These studies were designed to characterize the complex changes in Prdxs induced by oxidant stress in rat myocardium. Hydrogen peroxide, a Prdx substrate, was used as the model oxidant pertinent to redox signaling during health and to injury at higher concentrations. Rat hearts were aerobically perfused with a broad concentration range of hydrogen peroxide by the Langendorff method, homogenized, and analyzed by immunoblotting. Heart extracts were also analyzed by size-exclusion chromatography under nondenaturing conditions. Hydrogen peroxide-induced changes in disulfide bond formation, nonreversible oxidation of cysteine (hyperoxidation), and subcellular localization were determined. Hydrogen peroxide induced an array of changes in the myocardium, including formation of disulfide bonds that were intermolecular for Prdx1, Prdx2, and Prdx3 but intramolecular within Prdx5. For Prdx1, Prdx2, and Prdx5, disulfide bond formation can be approximated to an EC50 of 10–100, 1–10, and 100–1,000 μM peroxide, respectively. Hydrogen peroxide induced hyperoxidation, not just within monomeric Prdx (by SDS-PAGE), but also within Prdx disulfide dimers, and reflects a flexibility within the dimeric unit. Prdx oxidation was also associated with movement from the cytosolic to the membrane and myofilament-enriched fractions. In summary, Prdxs undergo a complex series of redox-dependent structural changes in the heart in response to oxidant challenge with its substrate hydrogen peroxide.

Download Full-text

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers

Biochemical Journal ◽

10.1042/bj20101396 ◽

2011 ◽

Vol 434 (2) ◽

pp. 219-231 ◽

Cited By ~ 15

Author(s):

Jörg Behnke ◽

Eeva-Liisa Eskelinen ◽

Paul Saftig ◽

Bernd Schröder

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Transmembrane Protein ◽

Bond Formation ◽

Immunogold Labelling ◽

Disulfide Bridge ◽

Cysteine Residue ◽

Disulfide Bond Formation ◽

Transmembrane Segments ◽

Late Endosomes

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.

Download Full-text