Intersubunit Disulfide Bond Research Articles

Physcomitrium patens flavodiiron proteins form heterotetrametric complexes

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.

Zinc transporter 1 functions in copper uptake and cuproptosis

Copper (Cu) is a co-factor for several essential metabolic enzymes. Disruption of Cu homeostasis results in genetic diseases such as Wilson's disease. Here, we show that the zinc transporter 1 (ZnT1), known to export zinc (Zn) out of the cell, also mediates Cu2+ entry into cells and is required for Cu2+-induced cell death, cuproptosis. Structural analysis and functional characterization indicate that Cu2+ and Zn2+ share the same primary binding site, allowing Zn2+ to compete for Cu2+ uptake. Among ZnT members, ZnT1 harbors a unique inter-subunit disulfide bond that stabilizes the outward-open conformations of both protomers to facilitate efficient Cu2+ transport. Specific knockout of the ZnT1 gene in the intestinal epithelium caused the loss of Lgr5+ stem cells due to Cu deficiency. ZnT1, therefore, functions as a dual Zn2+ and Cu2+ transporter and potentially serves as a target for using Zn2+ in the treatment of Wilson's disease caused by Cu overload.

Unlocking the gelling potential of oat protein: Synergistic effects of sonication and disulfide cleavage

The high thermal stability of oat globulin, attributed to inter-subunit disulfide bonds and strong hydrophobic interactions, poses a challenge to its gelling capacity. This study investigated the heat-induced gelling properties of oat protein isolate (OPI), focusing on the synergistic effect of physical (high-intensity ultrasound, HIU) and chemical (cysteine-assisted disulfide bond disruption) treatments. Creep-recovery and viscoelasticity (Gʹ/Gʹʹ) tests revealed decreased deformability and enhanced rigidity of OPI intermolecular structure upon cysteine treatment (100–400 mg/g protein), especially for HIU-treated protein. The HIU + cysteine process substantially increased the gel hardness (from 0.005 to 0.69 N) over the individual treatments with a synergy up to 432%. Correspondingly, cooking loss was reduced by 28–98% due to the move of free water into a restricted state (protein-bound protons) within the gel matrix as detected by 1H NMR. These findings suggest new possibilities for utilizing oat protein in gel-based food products.

Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR)

The most common cardiac pathologies, such as myocardial infarction and heart failure, are associated with oxidative stress. Oxidation of the cardiac ryanodine receptor (RyR2) Ca2+ channel causes spontaneous oscillations of intracellular Ca2+, resulting in contractile dysfunction and arrhythmias. RyR2 oxidation promotes the formation of disulfide bonds between two cysteines on neighboring RyR2 subunits, known as intersubunit cross-linking. However, the large number of cysteines in RyR2 has been a major hurdle in identifying the specific cysteines involved in this pathology-linked post-translational modification of the channel. Through mutagenesis of human RyR2 and in-cell Ca2+ imaging, we identify that only two cysteines (out of 89) in each RyR2 subunit are responsible for half of the channel’s functional response to oxidative stress. Our results identify cysteines 1078 and 2991 as a redox-sensitive pair that forms an intersubunit disulfide bond between neighboring RyR2 subunits during oxidative stress, resulting in a pathological “leaky” RyR2 Ca2+ channel.

Up-regulated oxidized USP2a can increase Mdm2-p60-p53 to promote cell apoptosis

Mdm2 promotes the ubiquitination and degradation of p53, while Mdm2-p60 can bind to p53 and reduce the Mdm2-induced p53 ubiquitination to improve its stability. USP2a can deubiquitinate and stabilize Mdm2, whether USP2a can regulate Mdm2-p60 needs to be further confirmed and elucidated. We found that oxidative stress can up-regulate USP2a at the post-transcriptional level and induce USP2a to be oxidized by forming inter-subunit disulfide bonds. The oxidized USP2a is closely related with cell apoptosis. In apoptotic cells, oxidized USP2a has enhanced protein stability and further stabilizes Mdm2-p60 through deubiquitination, and the USP2a-Mdm2-p60-p53 axis plays a role in cell apoptosis. Altogether USP2a is oxygen sensitive, oxidized USP2a exerts apoptotic effects through the Mdm2-p60-p53 axis, which provides an experimental basis for regulating p53 apoptotic signaling by targeting USP2a.

Intersubunit cross-linking is the key mechanism of cardiac ryanodine receptor dysfunction during oxidative stress: The role of cysteines 1078 and 2991.

The type 2 ryanodine receptor (RyR2) plays a key role in regulating cardiac intracellular Ca2+. We have previously shown that oxidative stress can regulate RyR2 by promoting the formation of disulfide bonds between neighboring RyR2 subunits (intersubunit cross-linking). However, the functional significance of this redox modification and cysteines involved in cross-linking remain unknown. Here, we used site-directed mutagenesis to replace cysteines 1078 and 2991 with serines in human RyR2 (hRyR2). Obtained constructs were expressed in HEK293 cells and treated with diamide to induce oxidative stress. Western blot analysis revealed that both mutations substantially protected against hRyR2 cross-linking. Analysis of the ER lumen [Ca2+] ([Ca2+]ER) dynamics with the ER Ca2+ sensor R-CEPIA1er in HEK293 cells expressing hRyR2 revealed that the mutation of either 1078 or 2991 cysteines prevented the depletion of ER Ca2+ load caused by RyR2 oxidation (by >40%). The simultaneous mutation of both cysteines produced the similar protection against hRyR2 cross-linking and [Ca2+]ER depletion, indicating a common mechanism of redox regulation involving both residues. Given the structural localization of both cysteines at the neighboring RyR2 subunits, we suggest that they form an intersubunit disulfide bond during channel oxidation, followed by the formation of the pathologically “leaky” channel. Although the N-terminal region (NTR) of RyR2 plays an important role in the channel's tetramer formation and functional regulation, the individual cysteines in this region are not directly involved in hRyR2 cross-linking. However, the structural integrity of the NTR has an allosteric effect on hRyR2 complex re-arrangements required for the channel's cross-linking. In conclusion, the results of this study revealed two critical cysteines within hRyR2 that are involved in the intersubunit disulfide cross-linking and the channel's functional response to oxidative stress.

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering.The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c, and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit.BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme.We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373.Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three β-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Probing the Dynamics of AcrB Through Disulfide Bond Formation.

The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.

Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.

Probing the Homo- and Hetero-Dimerization Propensities of Metabotropic Glutamate Receptors

The metabotropic glutamate receptors (mGluR) are class C G-protein coupled receptors (GPCR) which play important roles in modulating excitatory and inhibitory synaptic transmission. mGluRs function as dimers which are covalently linked through an intersubunit disulfide bond located in the ligand binding domain. In recent years it has become clear that some mGluR combinations can form hetero-dimers with distinct functional properties. However, little is known about the relative propensities for homo- versus hetero-dimerization and the mechanisms that regulate specific mGluR assembly. Here, we develop a quantitative, fluorescence-based complementation assay to study dimerization of mGluRs in living cells. We find a range of dimerization propensities, including many cases of preferential hetero-dimerization compared to homo-dimerization. Single molecule pulldown of full-length mGluR2 confirms the relative propensities which includes a strong preference for mGluR2/3 heterodimerization, which we further validate with native proteins. Finally, using a combination of structural and computational analysis in conjunction with fluorescence-based studies we probe the role of relative conformational dynamics and inter-subunit interface complementarity in determining dimerization specificity. Overall, our findings support the biological relevance of mGluR heterodimerization and motivate a thorough analysis of the physiological roles of different dimer combinations.

Probing the Dynamic Aspects of AcrB Function through Disulfide Bond Formation

The Resistant Nodulation Division (RND) super family member, tripartite complex AcrA-AcrB-TolC efflux pump is one of the major pumps contributing to the multidrug-resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux pump that is responsible for the recognition and binding of compounds before transportation out of the cell. Understanding the dynamics of AcrB regarding the functional rotation of the monomers in a trimeric functional unit in the process of efflux of drugs is the focus of this study. For this purpose, we created 7 disulfide bond pairs in the periplasmic domain of the AcrB using site directed mutagenesis to study the rotation and flexibility of the monomers. These pairs were chosen to be in close proximity to form inter-subunit disulfide bonds. Western blot analysis revealed the formation of disulfide oligomers under non reducing condition and monomers formation under reducing condition. Furthermore, minimum inhibitory concentration assays revealed the difference in antibiotic susceptibility of single and double Cys mutation in which the majority of double Cys mutation have a higher susceptibility to drugs, supporting that disulfide bond formation has facilitate the reduction of efflux activity. This was further confirmed with EtBr accumulation assays utilizing DTT as the reducing agent. In some cases, the activity of the double Cys-mutations was partially or completely restored by DTT, confirming the deleterious effect of the corresponding disulfide bond on protein activity. These findings lead to new insight in conformational changes critical for the activity of the dynamic AcrA-AcrB-TolC efflux pump in E.coli.

SOS and IP Modifications Predominantly Affect the Yield but Not Other Properties of SOSIP.664 HIV-1 Env Glycoprotein Trimers.

Soluble recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an intersubunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41ECTO) subunits and a point substitution, I559P (IP), to further stabilize the gp41ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP change can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have properties highly comparable to those of the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell but have little impact on the properties of purified trimers.

The melanocortin-1 receptor reversely regulates the melanin synthesis and migration of melanoma cells via dimerization-induced conformational changes

We previously reported that the melanocortin-1 receptor (MC1R), a key regulator of melanogenesis, regulates cell migration; however, the detailed mechanism remained unknown. Since the homo-dimerization of MC1R by four inter-subunit disulfide bonds is known to be functionally important for melanogenesis, we investigated the importance of MC1R dimerization for cell migration. Unlike the wild-type MC1R, the dimerization-defective mutant MC1R in which four critical Cys residues were replaced with Ala residues (Cys35-267-273-275Ala) significantly inhibited melanin synthesis but enhanced cell migration in human MNT-1 and A375 melanoma cells. This suggests that there may be a reverse correlation between melanin synthesis and cell migration. Interestingly, melanoma cells expressing the dimerization-defective mutant exhibited enhanced expression of the cell surface heparan sulfate proteoglycan, syndecan-2, and knockdown of syndecan-2 expression decreased the mutant-mediated cell migration. Consistently, ASIP, an antagonist of MC1R, enhanced syndecan-2 expression and cell migration and reversed the α-melanocyte-stimulating hormone (α-MSH)-mediated inhibition of syndecan-2 expression. Furthermore, α-MSH reduced the cell migration of MNT1 cells expressing wild-type MC1R but not its dimerization-defective mutant. Together, these data strongly suggest that MC1R reversely regulates melanin synthesis and migration via the conformational changes induced by dimerization.

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.

Can passive immunization prevent disease outcome in gilthead sea bream exposed to Enteromyxum leei?

IL-12 is an important cytokine that connects the innate and adaptive immune systems. The complete gene structure of olive flounder IL-12 and its characteristics have not yet been formally reported. Here, we report the complete sequences of both subunits of olive flounder IL-12 (IL-12p35 and IL-12p40). In addition, its function was analyzed by generating the single-chain rIL-12 of which subunits were fused by a GS linker and the rIL-12-specific mouse antibody. The cDNA sequences of IL-12p35 and IL-12p40 were 1059 nucleotides and 1319 nucleotides, respectively. The analyses of their gene structures, deduced amino acid sequences, protein model structures, and phylogenetic trees confirmed the accurate identification of olive flounder IL-12. The protein structure model suggested that an inter-subunit disulfide bond might be formed between the Cys177 of p35 and Cys74 of p40 to link the subunits. Olive flounder expressed IL-12p40 at higher levels than IL-12p35 in the various tissues under natural conditions although both expression levels were low. However, when infected by Edwardsiella tarda or stimulated by LPS, the flounder expressed both of the subunit genes at similar maximized levels in 6 h and gradually reduced thereafter. Olive flounder PBMC induced with the rIL-12 increased IFN-γ and TNF-α expression but decreased IL-10 expression as did treatment with LPS. However, when the LPS-treated PBMC were neutralized with the rIL-12-specific antibody, the pattern of cytokine expression was precisely reversed. In conclusion, we have formally identified the gene structure and function of olive flounder IL-12.

Impact of high temperature on the innate immune response in the intestine of orange-spotted grouper

IL-12 is an important cytokine that connects the innate and adaptive immune systems. The complete gene structure of olive flounder IL-12 and its characteristics have not yet been formally reported. Here, we report the complete sequences of both subunits of olive flounder IL-12 (IL-12p35 and IL-12p40). In addition, its function was analyzed by generating the single-chain rIL-12 of which subunits were fused by a GS linker and the rIL-12-specific mouse antibody. The cDNA sequences of IL-12p35 and IL-12p40 were 1059 nucleotides and 1319 nucleotides, respectively. The analyses of their gene structures, deduced amino acid sequences, protein model structures, and phylogenetic trees confirmed the accurate identification of olive flounder IL-12. The protein structure model suggested that an inter-subunit disulfide bond might be formed between the Cys177 of p35 and Cys74 of p40 to link the subunits. Olive flounder expressed IL-12p40 at higher levels than IL-12p35 in the various tissues under natural conditions although both expression levels were low. However, when infected by Edwardsiella tarda or stimulated by LPS, the flounder expressed both of the subunit genes at similar maximized levels in 6 h and gradually reduced thereafter. Olive flounder PBMC induced with the rIL-12 increased IFN-γ and TNF-α expression but decreased IL-10 expression as did treatment with LPS. However, when the LPS-treated PBMC were neutralized with the rIL-12-specific antibody, the pattern of cytokine expression was precisely reversed. In conclusion, we have formally identified the gene structure and function of olive flounder IL-12.

Analyses of the gene structure and function of olive flounder (Paralichthys olivaceus) interleukin 12 (IL-12)

IL-12 is an important cytokine that connects the innate and adaptive immune systems. The complete gene structure of olive flounder IL-12 and its characteristics have not yet been formally reported. Here, we report the complete sequences of both subunits of olive flounder IL-12 (IL-12p35 and IL-12p40). In addition, its function was analyzed by generating the single-chain rIL-12 of which subunits were fused by a GS linker and the rIL-12-specific mouse antibody. The cDNA sequences of IL-12p35 and IL-12p40 were 1059 nucleotides and 1319 nucleotides, respectively. The analyses of their gene structures, deduced amino acid sequences, protein model structures, and phylogenetic trees confirmed the accurate identification of olive flounder IL-12. The protein structure model suggested that an inter-subunit disulfide bond might be formed between the Cys177 of p35 and Cys74 of p40 to link the subunits. Olive flounder expressed IL-12p40 at higher levels than IL-12p35 in the various tissues under natural conditions although both expression levels were low. However, when infected by Edwardsiella tarda or stimulated by LPS, the flounder expressed both of the subunit genes at similar maximized levels in 6 h and gradually reduced thereafter. Olive flounder PBMC induced with the rIL-12 increased IFN-γ and TNF-α expression but decreased IL-10 expression as did treatment with LPS. However, when the LPS-treated PBMC were neutralized with the rIL-12-specific antibody, the pattern of cytokine expression was precisely reversed. In conclusion, we have formally identified the gene structure and function of olive flounder IL-12.

Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data

BackgroundProtein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs.ResultsApproximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging.ConclusionsCompared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.