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

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.

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

Cells ◽

10.3390/cells9091994 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1994 ◽

Cited By ~ 2

Author(s):

Philip J. Robinson ◽

Neil J. Bulleid

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Secretory Pathway ◽

Bond Formation ◽

Complex Problem ◽

Structure Stability ◽

Disulfide Bond Formation ◽

Polypeptide Folding ◽

Domains Of Life ◽

And Function

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.

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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.

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Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region

Biochemistry ◽

10.1021/bi00245a014 ◽

1991 ◽

Vol 30 (31) ◽

pp. 7766-7771 ◽

Cited By ~ 4

Author(s):

Akihiro Ishiwata ◽

Yasushi Kawata ◽

Kozo Hamaguchi

Keyword(s):

Disulfide Bond ◽

Light Chain ◽

Bond Formation ◽

Terminal Region ◽

Disulfide Bond Formation ◽

Immunoglobulin Light Chain

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Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus

Journal of General Virology ◽

10.1099/vir.0.068122-0 ◽

2014 ◽

Vol 95 (12) ◽

pp. 2820-2830 ◽

Cited By ~ 2

Author(s):

Feifei Yin ◽

Manli Wang ◽

Ying Tan ◽

Fei Deng ◽

Just M. Vlak ◽

...

Keyword(s):

Disulfide Bond ◽

Viral Entry ◽

Helicoverpa Armigera ◽

Disulfide Bonds ◽

Site Directed Mutagenesis ◽

Disulfonic Acid ◽

Viral Infectivity ◽

F Protein ◽

Free Thiol ◽

Helicoverpa Armígera

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4′-maleimidylstilbene 2,2′-disulfonic acid (AMS) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

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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.

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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.

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VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2015.09.017 ◽

2015 ◽

Vol 1848 (12) ◽

pp. 3188-3196 ◽

Cited By ~ 39

Author(s):

Masateru Okazaki ◽

Katsue Kurabayashi ◽

Miwako Asanuma ◽

Yohei Saito ◽

Kosuke Dodo ◽

...

Keyword(s):

Disulfide Bond ◽

Bond Formation ◽

Terminal Region ◽

Disulfide Bond Formation

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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.

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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.

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