Secreted MbovP0145 Promotes IL-8 Expression through Its Interactive β-Actin and MAPK Activation and Contributes to Neutrophil Migration

Mycoplasma bovis (M. bovis) is an important pathogen of cattle responsible for huge economic losses in the dairy and beef industries worldwide. The proteins secreted by M. bovis are mainly related to its adhesion, invasion, virulence, and intracellular survival and play a role in mycoplasma–host interactions. In our previous study, we found MbovP0145, a secreted protein present in the M. bovis secretome, but little is known about its function. In this study, we assessed the inflammatory characteristics and underlined mechanism of this inflammation of recombinant MbovP0145 (rMbovP0145). For this, bovine lung epithelial cells (EBL) were stimulated by rMbovP0145 to see the IL-8 production in a time- and dose-dependent manner. We observed that rMbovP0145 increased the production of IL-8 via ERK1/2 and P38 pathway activation. Further, the effect of the M. bovis ΔMbov_0145 mutant and its complementary strain on IL-8 mRNA expression was also confirmed. A pulldown assay of the GST-tagged MbovP0145 protein with mass spectrometry demonstrated that β-actin could specifically interact with rMbovP0145 to mediate the IL-8 signaling. As knockdown of β-actin expression with RNA interference in EBL cells decreased the mRNA expression of IL-8 and the phosphorylated ERK1/2 and P38 proteins, whereas disrupted actin polymerization by cytochalasin D led to a significantly higher IL-8 expression and MAPK phosphorylation in rMbovP0145-stimulated cells. Compared to M. bovis HB0801 and its complementary strain, the culture supernatant of EBL cells infected with the M. bovis ΔMbov_0145 mutant induced less neutrophil migration to the lower chamber in a transwell system. In conclusion, MbovP0145 promoted IL-8 expression by interacting with β-actin through activation of the MAPK pathway, thus contributing to neutrophil migration.

Mutations in CSRP3/MLP, a Z-disc associated gene are functionally associated with dilated cardiomyopathy in Indian population

CSRP3 is a LIM domain containing protein, known to play an important role in cardiomyocyte development, differentiation and pathology. Mutations in CSRP3 gene are reported in both dilated and hypertrophic cardiomyopathy (DCM and HCM), however, the genotype-phenotype correlation still remains elusive. To investigate the pathogenic potential of CSRP3 variants in our DCM cohort, we have screened 100 DCM cases and 100 controls and identified 3 non-synonymous variations, of which two are missense variants viz., c.233 GGC>GTC, p.G78V; c.420 TGG>TGC, p.W140C, and the third one is a single nucleotide polymorphism (SNP) c.46 ACC>TCC, p.T16S. These variants were absent from 100 control individuals (200 chromosomes). In vitro functional analysis has revealed reduction of CSRP3 protein level in stably-transfected C2C12 cells with p.G78V or p.W140C variants. Immunostaining demonstrates both cytoplasmic and nuclear localization of the wild-type protein, however variants p.G78V and p.W140C cause obvious reduction in the cytoplasmic expression of CSRP3 protein which is more pronounced in case of p.W140C. Disarrayed actin cytoskeleton was also observed in mutants. Besides, the expression of target genes namely Ldb3, Myoz2, Tcap, Tnni3 and Ttn are also downregulated in response to these variants. GST-pulldown assay has also showed a diminished binding of CSRP3 protein with α-Actinin due to both variants p.G78V and p.W140C. Both 2D, 3D-modeling have shown confirmational changes. Most in silico tools predict these variants as deleterious. Taken together, all these results suggest the impaired gene function due to these deleterious variants in CSRP3, implicating its possible disease causing role in DCM.

Cavin-1 modulates BMP/Smad signaling through the interaction of Caveolin-1 with BMPRII in pulmonary artery endothelial cells

Background Pulmonary hypertension (PH) is a progressive disease associated with poor outcomes. Caveolin-1 (Cav1) and Cavin-1 are components of caveolae, and Cav1 is identified as a related gene of pulmonary arterial hypertension (PAH). Gene mutations of bone morphogenetic protein type II receptor (BMPRII) is the most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Purpose Our study aims to investigate the relationship between Caveolin-Cavin system and BMP/Smad signaling pathway in pulmonary artery endothelial cells (PAECs). [Methods] Cav1 knockout mice were used to assess PH, and caveolae in PAECs were observed by electron microscope. After knocking down Cav1 and/or Cavin-1 in human PAECs (hPAECs) using siRNA, we evaluated the phosphorylation of Smad by Western blotting. Apoptosis was explored by flow cytometry. To assess the interaction between Cav1 and BMPRII, and the effect of Cavin-1 for this interaction and BMP/Smad signaling, we performed immunoprecipitation, Co-immunostaining, Proximal Ligation Assay (PLA), GST pulldown assay, and Western blotting. Results As in previous reports, Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy and PAECs isolated from Cav1 knockout mice showed caveolae disappearance. Cav1 knockdown in hPAECs reduced BMPRII at the plasma membrane and Smad 1/5/9 phosphorylation. Cav1 knockdown also significantly increased hypoxia-induced apoptosis in hPAECs. Co-immunostaining revealed that Cav1 was associated with BMPRII at the membrane of hPAECs. Cavin-1 inhibited the interaction of BMPRII with Cav1 and reduced BMPRII localization on the membrane of hPAECs. GST pulldown assay revealed that Cavin-1 and BMPRII were associated with Cav1 through the scaffolding domain in Cav1. These findings suggest that Cavin-1 and BMPRII are competitively associated with Cav1. Cavin-1 knockdown improved the interaction between Cav1 and BMPRII and inhibited both BMPRII reduction at the plasma membrane and Smad 1/5/9 dephosphorylation. Conclusions Cavin-1 affects the interaction of Cav1 with BMPRII at the plasma membrane and modulates BMP/Smad signaling in PAECs. The binding of Cavin-1 to Cav1 enhances the interaction between BMPR2 and Cav1, resulting in stabilization of BMPRII localization at the plasma membrane in PAECs and prevention of BMP/Smad signaling attenuation, which is important for PAH development. FUNDunding Acknowledgement Type of funding sources: None.

Chicken DDX1 Acts as an RNA Sensor to Mediate IFN-β Signaling Pathway Activation in Antiviral Innate Immunity

Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-β, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-β, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-β and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-β signaling pathways.

Improvement Lipophagy by Restoring Rab7 Cycle: Protective Effects of Quercetin on Ethanol-Induced Liver Steatosis

Background: Chronic alcohol consumption induces lipophagy retard which contributes the pathogenesis of liver steatosis. Lipophagy-related Rab7 responsible for the fusion between autophagosomes and lysosomes has been presumed as a crucial regulator on the progression of alcohol liver disease (ALD) despite elusive mechanisms. More importantly, whether hepatoprotective quercetin targets Rab7-associated lipophagy disorder or not, still remains uncertain. Results: ALD mice were induced by chronic-plus-single-binge ethanol feeding that manifested by hampering autophagosomes formation with lipid droplets (LDs) and fuse with lysosomes compared with the normal control, which was normalized partially by quercetin. GST-RILP pulldown assay of Rab7 indicated an improved GTP-Rab7 as quercetin treatment for ethanol-feeding mice. Lipophagy was blocked when HepG2 cells were transfected with siRNA-Rab7, which was reversed through co-transfection of Rab7Wt plasmid. Rab7-specific inhibitor CID1067700 aggravated ethanol-induced steatosis and autophagic flux disruption visualized by immunofluorescence co-localization analysis and mCherry-GFP-LC3 transfection. HepG2 cells overexpressing Rab7Wt show a stronger alleviation on alcohol-induced lipophagy dysfunction than Rab7Q67L. Furthermore, TBC1D5 responsible for Rab7 inactivation was downregulated after alcohol administration, but regained by quercetin. Rab7 circulation retarded by ethanol and corrected by quercetin was further revealed by fluorescence recovery after photobleaching (FRAP). Conclusions: Chronic alcoholic not only inactivates Rab7 but also retards TBC1D5-mediated Rab7 turnover, synergistically obstructing lipophagy flux and promoting ethanol-induced steatosis. Quercetin attenuates adipohepatic degeneration by normalizing ethanol-imposed Rab7 disorders and subsequent lipophagy disturbance, highlighting a novel mechanism and promising prospect of quercetin-like phytochemicals against the crucial first hit from the alcohol.

Expression, Purification and in Silico Characterization of Mycobacterium Smegmatis Alternative Sigma Factor SigB

Sigma factor B (SigB), an alternative sigma factor (ASF) is very similar to primary sigma factor SigA (σ70) but dispensable for growth in both Mycobacterium smegmatis (Msmeg) and Mycobacterium tuberculosis (Mtb). It is involved in general stress responses including heat, oxidative, surface, starvation stress and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive Extra Cytoplasmic Function (ECF) sigma factors (SigH, SigE, SigL) and SigF involved in multiple signalling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of E. coli, viz; (BL21(DE3), C41(DE3) and CodonPlus(DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time-duration. We achieved the maximum expression of SigB at 25 °C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA is carried out using homology modelling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Mycobacterium smegmatis (Msmeg) and sigB. This effort provides a platform for pulldown assay, structural and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signalling networks along with its regulation.

DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein–protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus–host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21–FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

HALO-RPD: In Searching for RNA-Binding Protein Targets in Plants

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.

Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

Screening of the HBx transactivation domain interacting proteins and the function of interactor Pin1 in HBV replication

Hepatitis B virus (HBV) X protein (HBx) has been determined to play a crucial role in the replication and transcription of HBV, and its biological functions mainly depend on the interaction with other host proteins. This study aims at screening the proteins that bind to the key functional domain of HBx by integrated proteomics. Proteins that specifically bind to the transactivation domain of HBx were selected by comparing interactors of full-length HBx and HBx-D5 truncation determined by glutathione-S-transferase (GST) pull-down assay combined with mass spectrometry (MS). The function of HBx interactor Pin1 in HBV replication was further investigated by in vitro experiments. In this study, a total of 189 proteins were identified from HepG2 cells that specifically bind to the transactivation domain of HBx by GST pull-down and subsequent MS. After gene ontology (GO) analysis, Pin1 was selected as the protein with the highest score in the largest cluster functioning in protein binding, and also classified into the cluster of proteins with the function of structural molecule activity, which is of great potential to be involved in HBV life cycle. The interaction between Pin1 and HBx has been further confirmed by Ni2+-NTA pulldown assay, co-immunoprecipitation, and immunofluorescence microscopy. HBsAg and HBeAg levels significantly decreased in Pin1 expression inhibited HepG2.2.15 cells. Besides, the inhibition of Pin1 expression in HepG2 cells impeded the restored replication of HBx-deficient HBV repaired by ectopic HBx expression. In conclusion, our study identified Pin1 as an interactor binds to the transactivation domain of HBx, and suggested the potential association between Pin1 and the function of HBx in HBV replication.