Capping Protein Research Articles

Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include a Regulator of the Actin Cytoskeleton.

Transmembrane mucin-type glycoproteins can regulate signal transduction pathways. In yeast, signaling mucins regulate mitogen-activated protein kinase (MAPK) pathways that induce cell differentiation to filamentous growth (fMAPK pathway) and the response to osmotic stress (HOG pathway). To explore regulatory aspects of signaling mucin function, protein microarrays were used to identify proteins that interact with the cytoplasmic domain of the mucin-like glycoprotein Msb2p. Eighteen proteins were identified that comprised functional categories of metabolism, actin filament capping and depolymerization, aerobic and anaerobic growth, chromatin organization and bud growth, sporulation, ribosome biogenesis, protein modification by iron-sulfur clusters, RNA catabolism, and DNA replication and DNA repair. A subunit of actin capping protein, Cap2p, interacted with the cytoplasmic domain of Msb2p. Cells lacking Cap2p showed altered localization of Msb2p and increased levels of shedding of Msb2p's N-terminal glycosylated domain. Consistent with its role in regulating the actin cytoskeleton, Cap2p was required for enhanced cell polarization during filamentous growth. Our study identifies proteins that connect a signaling mucin to diverse cellular processes and may provide insight into new aspects of mucin function.

Identification and genetic characterization of porcine circovirus 3 on pig farms in Serbia.

The presence of PCV3 genome has been detected in pigs affected by different clinical and pathological conditions as well as in healthy animals. Its presence has been reported in many countries of North and South America, Asia and Europe. However, there is no evidence of the presence and genetic characteristics of PCV3 in many European countries and especially the countries of the Balkan Peninsula. The major objective of this study was to investigate the presence and obtain further genetic characterization of PCV-3 in the pig populations in Serbia. To demonstrate the presence of PCV-3 DNA a conventional PCR assay was performed. The samples where no PCR product was observed (n=32), were further tested with a real-time PCR assay. The six PCR samples that were strongly positive for PCV-3 were subjected to amplification and sequencing of their entire cap genes and complete viral genome. We report on the first identification, genetic diversity and potential association in pathogenesis of some systemic and respiratory swine diseases of PCV-3 in Serbia. The results imply that PCV-3 circulates widely in the pig population and has a high similarity with previously reported isolates. Detected PCV-3 can be associated with some swine systemic and respiratory diseases but these associations are strongly influenced by the clinical or pathological condition of the animals. Our findings demonstrate that there are certain PCV-3 loads in pigs suffering from active PRRSV infection, Glässer's disease, APP pleuropneumonia, pneumonic pasteurellosis and PRDC; however, the significance of this viral load, as well as the mechanism by which PCV-3 may act as a secondary agent in aggravating the severity during co-infections of these pathogens, requires further research.

Capping proteins regulate fungal development, DON-toxisome formation and virulence in Fusarium graminearum.

SummaryDeoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)‐actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.

Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.

Oral vaccination with the porcine circovirus type 2 (PCV-2) capsid protein expressed by Lactococcus lactis induces a specific immune response against PCV-2 in mice.

Porcine circovirus type 2 (PCV2) can cause postweaning, multisystemic wasting syndrome in pigs, which leads to enormous losses in the swine industry worldwide. Here, a genetically engineered Lactococcus strain expressing the main protective antigen of PCV2, the Cap protein, was developed to act against PCV2 infection as an oral vaccine. Expression of the Cap protein was confirmed via western blot, ELISA and fluorescence microscopy. Over 90% of the recombinant pAMJ399-Cap/MG1363 survived a simulated gastrointestinal transit. It also survived the murine intestinal tract for at least 11days. Then, the safety and immunogenicity of pAMJ399-Cap/MG1363 in orally immunized mice was evaluated. The levels of the sIgA, IgG and cytokines (IL-4 and IFN-γ) obtained from the mice immunized with pAMJ399-Cap/MG1363 were significantly higher than those in the control groups. pAMJ399-Cap/MG1363 can survive in the gastrointestinal transit and effectively induce mucosal, cellular and humoral immune response against PCV2 infection via oral administration. This study demonstrates the potential of the genetically engineered Lactococcus lactis as a candidate for an oral vaccine against PCV2.

Plant-based metal and metal alloy nanoparticle synthesis: a comprehensive mechanistic approach

There are enormous methods such as physical, chemical, and biological, for the synthesis of metallic nanoparticles (MNPs), which has become a matter of focus among material scientists. Green chemistry-based MNP synthesis is an area, which has gained much importance presently due to their non-toxicity and monodispersed nanoparticle preparation methodologies. Among green synthesis methods, plants are considered as efficient candidates for nanoparticle synthesis. The meticulous formation of different sizes and shapes of the nanoparticles using plants has spurred encouraging interest. The rate kinetics and stability of nanoparticle synthesis are well studied as well as appreciated in the arena of materials. Their capability to sequester metal ions and fastidiously define the dimensions using a plethora of capping proteins such as glutathione and phytochelatins is intriguing giving it a monodispersed size. This review is a comprehensive understanding of the metal nanoparticles synthesized by plants and apprehends the mechanism of nanoparticle synthesis exhaustively.

Metal ions and redox balance regulate distinct amyloid-like aggregation pathways of GAPR-1

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins.

Development of a simple and high-yielding fed-batch process for the production of porcine circovirus type 2 virus-like particle subunit vaccine

The cap protein is encoded by the orf2 gene of porcine circovirus type 2 (PCV2) has the main antigen epitope of PCV2 and can form virus-like particles (VLPs), which are expressed in insect cells. PCV2-VLPs can effectively inhibit PCV2 replication as a subunit vaccine. In this study, a robust and reliable fed-batch process was successfully developed for the production of PCV2-VLPs by Sf9 cells. The feeding solution, feeding strategy, and cell density at infection were optimized to maximize the final PCV2-VLPs production yields. The cell density at infection and the volumetric PCV2-VLPs production reached 12 × 106 cells/mL and 110 mg/L, respectively, which yielded 3- and 3.6-fold enhancements compared to the batch culture. The PCV2-VLPs produced in fed-batch culture were not different from the PCV2-VLPs produced in a batch culture in an immunity test. A highly efficient production process was produced for PCV2-VLPs subunit vaccines, which could provide an effective means for the industrial production of PCV2 vaccines.

Turnover versus treadmilling in actin network assembly and remodeling.

Actin networks are highly dynamic cytoskeletal structures that continuously undergo structural remodeling. One prominent way to probe these processes is via Fluorescence Recovery After Photobleaching (FRAP), which can be used to estimate the rate of turnover for filamentous actin monomers. It is thought that head-to-tail treadmilling and de novo filament nucleation constitute two primary mechanisms underlying turnover kinetics. More generally, these self-assembly activities are responsible for many important cellular functions such as force generation, cellular shape dynamics, and cellular motility. In what relative proportions filament treadmilling and de novo filament nucleation contribute to actin network turnover is still not fully understood. We used an advanced stochastic reaction-diffusion model in three dimensions, MEDYAN, to study turnover dynamics of actin networks containing Arp2/3, formin and capping protein at experimentally meaningful length- and time-scales. Our results reveal that, most commonly, treadmilling of older filaments is the main contributor to actin network turnover. On the other hand, although turnover and treadmilling are often used interchangeably, we show clear instances where this assumption would not be justified, for example, finding that rapid turnover is accompanied by slow treadmilling in highly dendritic Arp2/3 networks.

Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.

Generation and evaluation of a recombinant goose origin Newcastle disease virus expressing Cap protein of goose origin avastrovirus as a bivalent vaccine in goslings

Currently, both goose astrovirus (GoAstV) and goose-origin Newcastle disease virus (NDV) are widely infectious agents for goslings. There is no vaccine for GoAstV. Capsid protein can elicit a neutralizing antibody in human astroviruses (HAstV). Molecular analysis of the genomic region encoding the capsid protein(ORF2) of goose astrovirus has revealed that it contains neutralizing epitopes. Goose-origin NDV is also an infectious agent. A wide range of NDV strains exist that can be commonly used as vaccine vectors. In the present study, the fusion protein cleavage site RRQKR↓F in a backbone of the virulent goose-origin NDV SH-12 was changed into an avirulent motif GRQGR↓L. The modified goose-origin NDV recombinant vaccine virus expressing the Capsid protein (Cap) of GoAstV was generated as a bivalent vaccine using a reverse-genetics approach. The recombinant virus, rNDV/GoAstV-Cap, was attenuated and similar growth dynamics, cytopathic effects, and virus titers in vitro were maintained when compared to the LaSota strain. Expression of the GoAstV-Cap protein in rNDV/GoAstV-Cap infected cells was detected by an immunofluorescence assay and Western blotting. Goslings inoculated with rNDV/GoAstV-Cap showed no apparent signs of disease and induced GoAstV-Cap-specific immune responses and NDV-specific serum antibody responses to a LaSota vaccination control. Complete protection against a pathogenic GoAstV challenge and avelogenic NDV challenge was conferred. The results of the study suggested that rNDV/GoAstV-Cap viruses have the potential to be the safe, stable, and effective bivalent vaccines.

Quantitative protein detection using single molecule imaging enzyme-linked immunosorbent assay (iELISA)

Protein detection is a key step in molecular biology research and is required for pathogen and protein marker testing for disease diagnostics. Here, single molecule imaging enzyme-linked immunosorbent assay (iELISA) is proposed to quantitatively measure the porcine circovirus type 2 (PCV2) Cap protein. The monoclonal antibody against PCV2 Cap protein indirectly immobilized on a polyethylene glycol (PEG) passivated slide by biotin-streptavidin interaction is used to capture the PCV2 Cap protein, and the PCV2 Cap protein can be detected in single molecule level according to the fluorescein isothiocyanate (FITC)-labeled secondary antibody using total internal reflection fluorescence microscopy. The single molecule iELISA measurements can be finished within 1 h skipping the time-consuming sample preparation procedures; moreover, it also exhibits excellent protein selectivity and anti-interference capability. With the proposed single molecule iELISA, linear relation between the fluorescent signals and logarithm of target protein concentrations is obtained with the detection limit of 7 ng/mL. Considering its high accuracy in target protein detection with simple procedures and fast speed, it is believed single molecule iELISA can be potentially adopted in fast trace protein detection.

Identification and Characterization of a Distinct Strain of Beak and Feather Disease Virus in Southeast China.

Beak and feather disease virus (BFDV) is an infectious agent responsible for feather degeneration and beak deformation in birds. In March 2017, an epidemic of psittacine beak and feather disease (PBFD) struck a farm in Fuzhou in the Fujian Province of southeast China, resulting in the death of 51 parrots. In this study, the disease was diagnosed and the pathogen was identified by PCR and whole genome sequencing. A distinct BFDV strain was identified and named as the FZ strain. This BFDV strain caused severe disease symptoms and pathological changes characteristic of typical PBFD in parrots, for example, loss of feathers and deformities of the beak and claws, and severe pathological changes in multiple organs of the infected birds. Phylogenetic analysis showed that the FZ strain was more closely related to the strain circulating in New Caledonia than the strains previously reported in China. Nucleotide homology between the FZ strain and other 43 strains of BFDV ranged from 80.0% to 92.0%. Blind passage experiment showed that this strain had limited replication capability in SPF Chicken Embryos and DF-1 Cells. Furthermore, the capsid (Cap) gene of this FZ strain was cloned into the pGEX-4T-1 expression vector to prepare the polyclonal anti-Cap antibody. Western blotting analysis using the anti-Cap antibody further confirmed that the diseased parrots were infected with BFDV. In this study, a PBFD and its pathogen was identified for the first time in Fujian Province of China, suggesting that future surveillance of BFDV should be performed.

FPR1 is the plague receptor on host immune cells.

The plague agent, Yersinia pestis, employs a type III secretion system (T3SS) to selectively destroy human immune cells, thereby enabling its replication in the bloodstream and transmission to new hosts via fleabite. The host factors responsible for the selective destruction of immune cells by plague bacteria were not known. Here we show that LcrV, the needle cap protein of the Y. pestis T3SS, binds N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality, however N-formylpeptide receptor deficient animals exhibit increased survival and plague-protective antibody responses. We identified FPR1 p.R190W as a candidate human resistance allele that protects neutrophils from Y. pestis T3SS. These findings reveal the plague receptor on immune cells and show that FPR1 mutations provide for plague survival, which appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.

Porcine circovirus type II screening in feral swine population in Ukraine

Porcine circovirus type 2 (PCV2) is an emergent single-stranded DNA virus found all over the world in domestic pigs and wild boars that causes infectious disease with a great impact on swine productivity. PCV2 has 1.7 kb genome that includes two main genes, which encode replication-related protein (rep) and the major structural capsid (cap) protein. Both of these genes can be used as target sequences for the primer design for the detection of PCV2 as well as for sequencing of designated regions. We carried out a screening due to the PCV2 circulating among the wild boar population in 10 regions of Ukraine. PCR screening was performed using primer pairs designed on the target sequences of the replicative and capsid genes. According to the results of the research, the presence of genetic material of PCV2 was found in 31.8% of the tested samples. The developed set of primers may be suitable for diagnostics, as well as for the development of specific sites for the purpose of sequencing of PCV2 cap-gene due to the obtained DNA samples during epizootic screening

Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma Hyopneumoniae by a Baculovirus Expression System.

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.

Capping Protein Insulates Arp2/3-Assembled Actin Patches from Formins.

SummaryHow actin structures of distinct identities and functions coexist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits, and the formins For3, Cdc12, and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin, and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in cells lacking CP. CP deletion also rescues the fusion defect of a mutation in the Fus1 knob region. However, myosin V and exocytic cargoes are reduced at the fusion focus and diverted to ectopic foci, which underlies the fusion defect. Remarkably, the ectopic foci coincide with Arp2/3-assembled actin patches, which now contain low levels of Fus1. We further show that CP localization to actin patches is required to prevent the formation of ectopic foci and promote efficient cell fusion. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12, normally absent from patches, and are co-decorated by the linear filament-binding protein tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

Nanovaccine Confers Dual Protection Against Influenza A Virus And Porcine Circovirus Type 2.

BackgroundThe influenza A virus (IAV) is known for its high variability and poses a huge threat to the health of humans and animals. Pigs play a central role in the cross-species reassortment of IAV. Ectodomain of matrix protein 2 (M2e) is the most conserved protective antigen in IAV and can be used to develop nanovaccines through nanoparticles displaying to increase its immunogenicity. However, the high immunogenicity of nanoparticles can cause the risk of off-target immune response, and excess unwanted antibodies may interfere with the protective efficacy of M2e-specific antibodies. Therefore, it is necessary to select reasonable nanoparticles to make full use of antibodies against nanoparticles while increasing the level of M2e-specific antibodies. Porcine circovirus type 2 (PCV2) is the most susceptible virus in pigs and can promote IAV infection. It is meaningful to develop a vaccine that can simultaneously control swine influenza virus (SIV) and PCV2.MethodsIn the present study, M2e of different copy numbers were inserted into the capsid (Cap) protein of PCV2 and expressed in Escherichia coli to form self-assembled chimeric virus-like particles (VLPs) nanovaccine. BALB/c mice and pigs were immunized with these nanovaccines to explore optimal anti-IAV and anti-PCV2 immunity.ResultsCap is capable of carrying at least 81 amino acid residues (three copies of M2e) at its C-terminal without impairing VLPs formation. Cap-3M2e VLPs induced the highest levels of M2e-specific immune responses, conferring protection against lethal challenge of IAVs from different species and induced specific immune responses consistent with PCV2 commercial vaccines in mice. In addition, Cap-3M2e VLPs induced high levels of M2e-specific antibodies and PCV2-specific neutralizing antibodies in pigs.ConclusionCap-3M2e VLP is an economical and promising bivalent nanovaccine, which provides dual protection against IAV and PCV2.

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

A novel fluorescent sensor for cAMP based on E. coli CAP protein

A novel fluorescent sensor for cAMP based on E. coli CAP protein