Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

AbstractThe mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

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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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The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

Canadian Journal of Biochemistry ◽

10.1139/o79-035 ◽

1979 ◽

Vol 57 (3) ◽

pp. 279-285 ◽

Cited By ~ 1

Author(s):

Maire E. Percy ◽

Lebe Chang ◽

Catherine Demoliou ◽

Reuben Baumal

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Disulfide Bridge ◽

Myeloma Protein ◽

Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

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Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB

Infection and Immunity ◽

10.1128/iai.00938-17 ◽

2018 ◽

Vol 86 (5) ◽

Cited By ~ 6

Author(s):

Róisín M. McMahon ◽

Philip M. Ireland ◽

Derek S. Sarovich ◽

Guillaume Petit ◽

Christopher H. Jenkins ◽

...

Keyword(s):

Membrane Protein ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Burkholderia Pseudomallei ◽

Protein A ◽

Genomic Analysis ◽

Core Gene ◽

Content Type ◽

Redox Biology

ABSTRACT The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei . A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

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Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope

Methods and Protocols ◽

10.3390/mps1040040 ◽

2018 ◽

Vol 1 (4) ◽

pp. 40 ◽

Cited By ~ 4

Author(s):

Dalton Gibbs ◽

Anisa Kaur ◽

Anoja Megalathan ◽

Kumar Sapkota ◽

Soma Dhakal

Keyword(s):

Single Molecule ◽

Total Internal Reflection ◽

Life Sciences ◽

Inverted Microscope ◽

Sophisticated Instrument ◽

High Level ◽

Single Molecule Analysis ◽

Molecule Dynamics ◽

Vast Range

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.

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Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽

10.7554/elife.01434 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 84

Author(s):

Yann Gambin ◽

Nicholas Ariotti ◽

Kerrie-Ann McMahon ◽

Michele Bastiani ◽

Emma Sierecki ◽

...

Keyword(s):

Single Molecule ◽

Self Assembly ◽

Mammalian Cells ◽

Protein Complexes ◽

Building Blocks ◽

Scaffolding Protein ◽

Cell Extracts ◽

Single Molecule Analysis ◽

Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

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Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1

Journal of Biological Chemistry ◽

10.1074/jbc.m704465200 ◽

2007 ◽

Vol 282 (38) ◽

pp. 28087-28095 ◽

Cited By ~ 113

Author(s):

Jun-ichi Niwa ◽

Shin-ichi Yamada ◽

Shinsuke Ishigaki ◽

Jun Sone ◽

Miho Takahashi ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Familial Amyotrophic Lateral Sclerosis ◽

Neuron Death ◽

Mutant Sod1 ◽

Intermolecular Disulfide Bonds ◽

High Molecular Weight Aggregate ◽

Lateral Sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6- and Cys111-disulfide cross-linked form and targeted it for proteasomal degradation.

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Folic Acid Decorated β-Cyclodextrin-Based Poly(ε-Caprolactone)/Dextran Star Polymer with Disulﬁde Bond-Linker as Theranostic Nanoparticle for Tumor-Targeted MRI and Chemotherapy

10.20944/preprints202110.0196.v1 ◽

2021 ◽

Author(s):

Huikang Yang ◽

Nianhua Wang ◽

Ruimeng Yang ◽

Li-Ming Zhang ◽

Xinqing Jiang

Keyword(s):

Folic Acid ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Folate Receptor ◽

Superparamagnetic Iron Oxide ◽

Star Polymer ◽

Iron Oxide Particles ◽

Theranostic Nanoparticles ◽

Targeted Mri

β-cyclodextrin-based star polymers have attracted much interest because of their unique structures and potential biomedical and biological applications. Herein, we synthesized well-defined folic acid (FA)-conjugated and disulfide bond-linked star polymer ((FA-Dex-SS)-βCD-(PCL)14) acted as theranostic nanoparticles for tumor-targeted magnetic resonance imaging (MRI) and chemotherapy. Theranostic nanoparticles were obtained by loading doxorubicin (DOX) and superparamagnetic iron oxide particles (SPIO) were loaded into the star polymer nanoparticles to obtain ((FA-Dex-SS)-βCD-(PCL)14@DOX/SPIO) theranostic nanoparticles. In vitro drug release studies showed that approximately 100% of the DOX was released from disulfide bond-linked theranostic nanoparticles within 24 h under a reducing environment in the presence of 10.0 mM GSH. DOX and SPIO could be delivered into HepG2 cells efficiently, owing to folate receptor-mediated endocytosis process of the nanoparticles and GSH triggered disulfide-bonds cleaving.Moreover, (FA-Dex-SS)-βCD-(PCL)14@DOX/SPIO showed strong MRI contrast enhancement properties. In conclusion, folate-decorated reduction-sensitive star polymeric nanoparticles are a potential theranostic nanoparticle candidate for tumor-targeted MRI and chemotherapy.

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Folic Acid-Decorated β-Cyclodextrin-Based Poly(ε-caprolactone)-dextran Star Polymer with Disulfide Bond-Linker as Theranostic Nanoparticle for Tumor-Targeted MRI and Chemotherapy

Pharmaceutics ◽

10.3390/pharmaceutics14010052 ◽

2021 ◽

Vol 14 (1) ◽

pp. 52

Author(s):

Huikang Yang ◽

Nianhua Wang ◽

Ruimeng Yang ◽

Liming Zhang ◽

Xinqing Jiang

Keyword(s):

Folic Acid ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Polymeric Nanoparticles ◽

Folate Receptor ◽

Superparamagnetic Iron Oxide ◽

Star Polymer ◽

Theranostic Nanoparticles ◽

Targeted Mri

β-cyclodextrin(βCD)-based star polymers have attracted much interest because of their unique structures and potential biomedical and biological applications. Herein, a well-defined folic acid (FA)-conjugated and disulfide bond-linked star polymer ((FA-Dex-SS)-βCD-(PCL)14) was synthesized via a couple reaction between βCD-based 14 arms poly(ε-caprolactone) (βCD-(PCL)14) and disulfide-containing α-alkyne dextran (alkyne-SS-Dex), and acted as theranostic nanoparticles for tumor-targeted MRI and chemotherapy. Theranostic nanoparticles were obtained by loading doxorubicin (DOX), and superparamagnetic iron oxide (SPIO) particles were loaded into the star polymer nanoparticles to obtain ((FA-Dex-SS)-βCD-(PCL)14@DOX-SPIO) theranostic nanoparticles. In vitro drug release studies showed that approximately 100% of the DOX was released from disulfide bond-linked theranostic nanoparticles within 24 h under a reducing environment in the presence of 10.0 mM GSH. DOX and SPIO could be delivered into HepG2 cells efficiently, owing to the folate receptor-mediated endocytosis process of the nanoparticles and glutathione (GSH), which triggered disulfide-bonds cleaving. Moreover, (FA-Dex-SS)-βCD-(PCL)14@DOX-SPIO showed strong MRI contrast enhancement properties. In conclusion, folic acid-decorated reduction-sensitive star polymeric nanoparticles are a potential theranostic nanoparticle candidate for tumor-targeted MRI and chemotherapy.

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Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

The Journal of Cell Biology ◽

10.1083/jcb.201512016 ◽

2016 ◽

Vol 214 (6) ◽

pp. 705-718 ◽

Cited By ~ 32

Author(s):

Ye Jin Chai ◽

Emma Sierecki ◽

Vanesa M. Tomatis ◽

Rachel S. Gormal ◽

Nichole Giles ◽

...

Keyword(s):

Single Molecule ◽

Molecular Chaperone ◽

Neurotransmitter Release ◽

Lewy Body ◽

Epileptic Encephalopathy ◽

Chaperone Activity ◽

Wild Type ◽

Developmental Defects ◽

Single Molecule Analysis

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

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Per Os Infectivity Factor 5 Identified as a Substrate of P33 in the Baculoviral Disulfide Bond Formation Pathway

Journal of Virology ◽

10.1128/jvi.00615-20 ◽

2020 ◽

Vol 94 (15) ◽

Author(s):

Huanyu Zhang ◽

Wenhua Kuang ◽

Cheng Chen ◽

Yu Shang ◽

Xiaoyan Ma ◽

...

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Bond Formation ◽

Oral Infection ◽

Progeny Virus ◽

Disulfide Bond Formation ◽

Sulfhydryl Oxidase ◽

Dna Viruses ◽

Formation Pathway

ABSTRACT Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection. IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulﬁde-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.

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