Glutathione S-transferase Pull-down Assays Research Articles

Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening.

Chinese sacbrood virus (CSBV) is the major cause and lead to the collapse of Apis cerana colonies. VP1, the structural protein of CSBV, shows the highest variation in the amino acid sequences among proteins from different CSBV strains as well as exhibits excellent immunogenicity. However, its function with host protein still remains unclear. To clarify its function with host protein, we screened out host cellular proteins that interact with VP1 using the membrane protein yeast two-hybrid system. In addition, we verified interactions between heat shock protein 70 cognate 5 (Hsp70-c5) and VP1 using glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. VP1 and Hsp70-c5 were colocalized in the cytoplasm and nucleus. Using western blot and real-time polymerase chain reaction (PCR), Hsp70-c5 expression in CSBV-infected larvae was upregulated compared with that in healthy larvae. We observed that when we silenced Hsp70-c5, VP1 expression was significantly downregulated. These results demonstrate that Hsp70-c5 is involved in at least one stage(s) of the viral life cycle.

Novel Interactions between the Human T-Cell Leukemia Virus Type 1 Antisense Protein HBZ and the SWI/SNF Chromatin Remodeling Family: Implications for Viral Life Cycle.

The human T-cell leukemia virus type 1 (HTLV-1) regulatory proteins Tax and HBZ play indispensable roles in regulating viral and cellular gene expression. BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, has been demonstrated to be essential not only for Tax transactivation but also for viral replication. We sought to investigate the physical interaction between HBZ and BRG1 and to determine the effect of these interactions on Tax-mediated long terminal repeat (LTR) activation. We reveal that HTLV-1 cell lines and adult T-cell leukemia (ATL) cells harbor high levels of BRG1. Using glutathione S-transferase (GST) pulldown and coimmunoprecipitation assays, we have demonstrated physical interactions between BRG1 and HBZ and characterized the protein domains involved. Moreover, we have identified the PBAF signature subunits BAF200 and BAF180 as novel interaction partners of HBZ, suggesting that the PBAF complex may be required for HTLV-1 transcriptional repression by HBZ. Additionally, we found that BRG1 expression translocates HBZ into distinct nuclear foci. We show that HBZ substantially represses HTLV-1 LTR activation by Tax/BRG1. Interestingly, we found that Tax stabilizes the expression of exogenous and endogenous BRG1 and that HBZ reverses this effect. Finally, using a chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay, we illustrate that HBZ facilitates the downregulation of HTLV-1 transcription by deregulating the recruitment of SWI/SNF complexes to the promoter. Overall, we conclude that SWI/SNF complexes, in addition to other cellular transcription factors, are involved in HBZ-mediated suppression of HTLV-1 viral gene expression.IMPORTANCE The pathogenic potential of HTLV-1 is linked to the indispensable multifaceted functions of the viral regulatory proteins Tax and HBZ, encoded by the sense and antisense viral transcripts, respectively. The interaction between Tax and the SWI/SNF family of chromatin remodeling complexes has been associated with HTLV-1 transcriptional activation. To date, the relationship between the SWI/SNF chromatin remodeling family and HBZ, the only viral protein that is consistently expressed in infected cells and ATL cells, has not been elucidated. Here, we have characterized the biological significance of the SWI/SNF family in regard to viral transcriptional repression by HBZ. This is important because it provides a better understanding of the function and role of HBZ in downregulating viral transcription and, hence, its contribution to viral latency and persistence in vivo, a process that may ultimately lead to the development of ATL.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

Purification of insoluble GST-fused and GST-cleaved Cav1.2 channel fragment by denaturation and renaturation

Both recombinant glutathione-S-transferase (GST)-fused and GST-cleaved fragments of an L-type voltage-gated Ca2+ channel (Cav1.2) are used frequently in GST pull-down assays to investigate the interactions between regulatory proteins and the Cav1.2 channel. However, GST-fused and GST-cleaved proximal C-terminal fragments of the guinea-pig cardiac Cav1.2 channel (CT1, amino acids 1509–1791) heterologously expressed in Escherichia coli (E. coli) are difficult to be recovered in a bioactive form because they are only poorly soluble. In this study, we developed a new method to solubilize and purify CT1. GST–CT1 expressed in E. coli was extracted and treated with an inclusion body solubilization and renaturation kit. Then, after adsorption to glutathione Sepharose beads, GST–CT1 was treated with protease to release CT1. However, the cleaved CT1 was insoluble and remained attached to the beads. Therefore, CT1 was treated again with the inclusion body solubilization and renaturation kit. Using this method, GST-CT1 and CT1 were purified with a high yield. GST pull-down experiments showed a dose-dependent interaction between GST–CT1 and calmodulin (CaM), and between GST–CaM and CT1, suggesting recovered bioactivity of GST–CT1 and CT1. This protocol may also be applied to purify other insoluble GST-fused proteins.

Lgr5-mediated p53 Repression through PDCD5 leads to doxorubicin resistance in Hepatocellular Carcinoma

The devastating prognosis of hepatocellular carcinoma (HCC) is partially attributed to chemotherapy resistance. Accumulating evidence suggests that the epithelial-mesenchymal transition (EMT) is a key driving force of carcinoma metastasis and chemoresistance in solid tumors. Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), as an EMT inducer, is involved in the potentiation of Wnt signaling in HCC. This study proposes uncovering the roles of Lgr5 in Doxorubicin (Dox) resistance of HCC to improve treatment efficacy for HCC.Methods: We investigated the expression and significance of Lgr5 in HCC tissue and different cell lines. The effect of Lgr5 in EMT and Dox resistance was analyzed in HCC cells and implanted HCC tumor models. A two-hybrid analysis, using the Lgr5 gene as the bait and a HCC cDNA library, was used to screen targeted proteins that interact with Lgr5. The positive clones were identified by coimmunoprecipitation (Co-IP) and Glutathione-S-transferase (GST) pull-down. The impact of the interaction on Dox resistance was investigated by a series of assays in vitro and in vivo .Result: We found that Lgr5 was upregulated and positively correlated with poor prognosis in HCC. Additionally, it functioned as a tumor promoter to increase cell migration and induce EMT in HCC cells and increase the resistance to Dox. We identified programmed cell death protein 5 (PDCD5) as a target gene of Lgr5 and we found that PDCD5 was responsible for Lgr5-mediated Dox resistance. Further analysis with Co-IP and GST pull-down assays showed that the N-terminal extracellular domain of Lgr5 could directly bind to PDCD5. Lgr5 induced p53 degradation by blocking the nuclear translocation of PDCD5 and leading to the loss of p53 stabilization. Lgr5 showed a protection against the inhibition of Dox on the growth of tumor subcutaneously injected. Moreover, Lgr5 suppressed Dox-induced apoptosis via the p53 pathway and attenuated the cytotoxicity of Dox to HCC.Conclusion: Lgr5 induces the EMT and inhibits apoptosis, thus promoting chemoresistance by regulating the PDCD5/p53 signaling axis. Furthermore, Lgr5 may be a potential target gene for overcoming Dox resistance.

Sertad1 promotes prostate cancer progression through binding androgen receptor ligand binding domain.

Androgen receptor (AR) signaling is involved in the initiation and progression of prostate cancer (PCa), which is the most frequently diagnosed nonskin cancer and remains a leading cause of cancer-related death in men. Further investigation of the involvement of AR signaling in PCa progression is urgently needed. In the present study, we performed a yeast two-hybrid screen and demonstrated that SERTA domain-containing protein 1 (Sertad1) is a novel AR-binding protein that binds to the AR ligand binding domain (LBD). The binding between AR-LBD and Sertad1 was confirmed by glutathione S-transferase (GST) pull-down assays and immunoprecipitation (IP) and confocal immunofluorescence co-localization experiments. Furthermore, we demonstrated that DHT inhibited Sertad1 protein degradation in prostate cancer cell lines and that Sertad1 knockdown inhibited the proliferation of prostate cancer cells in vitro. In human PCa tumor tissues, Sertad1 expression is positively correlated with AR expression and the Gleason score. Taken together, this report is the first to show that Sertad1 is a novel AR-LBD-binding protein, and DHT-liganded AR-LBD inhibits Sertad1 degradation. Thus, Sertad1 may represent a novel therapeutic target for the treatment of AR-positive PCa.

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.

Classical swine fever virus non-structural protein 4B binds tank-binding kinase 1

Classical swine fever (CSF) is a contagious disease with a high mortality rate and is caused by classical swine fever virus (CSFV). CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However, precisely how NS4B exerts these functions remains unknown, especially as there are no reports relating to potential cellular partners of CSFV NS4B. Here, a yeast two-hybrid (Y2H) system was used to screen the cellular proteins interacting with NS4B from a porcine alveolar macrophage (PAM) cDNA library. The protein screen along with alignment using the NCBI database revealed 14 cellular proteins that interact with NS4B: DDX39B, COX7C, FTH1, MAVS, NR2F6, RPLP1, PSMC4, FGL2, MKRN1, RPL15, RPS3, RAB22A, TP53BP2 and TBK1. These proteins mostly relate to oxidoreductase activity, signal transduction, localization, biological regulation, catalytic activity, transport and metabolism by GO categories. Tank-binding kinase 1 (TBK1) was chosen for further confirmation. The NS4B-TBK1 interaction was further confirmed by subcellular co-location, co-immunoprecipitation and glutathione S-transferase pull-down assays. This study offers a theoretical foundation for further understanding of the diversity of NS4B functions in relation to viral infection and subsequent pathogenesis.

Integrin β1 promotes gemcitabine resistance in pancreatic cancer through Cdc42 activation of PI3K p110β signaling

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignancies with very poor prognosis due to its broad resistance to chemotherapy. Our previous study showed that integrin β1 expression is upregulated in PDAC and confers gemcitabine resistance in PDAC cells via the signaling pathway including Cdc42 and AKT activation. But the accurate signal transductions are not clear. Here, we aimed to illuminate the signal transductions of integrin β1 in the acquisition of gemcitabine resistance in PDAC. Drug-resistance (DR) cells from AsPC-1 parent cell line (PCL) were selected. Integrin β1 expression was determined using western blot assay. Changes in drug response and the activity of phosphatidylinositol 3-kinase (PI3K) signaling after knockdown of integrin β1, Cdc42 or p110β were evaluated using MTT, cleaved caspase-3 immunofluorescence and western blot assay. Western blot assays also detected the variations in Cdc42 activity and p110β expression after integrin β1 knockdown. The interaction between Cdc42 and p110β was determined by Glutathione S-transferase (GST) pull-down assay. The results showed that integrin β1 expression was upregulated in DR-AsPC-1 cells, and integrin β1 knockdown significantly decreased the activity of Cdc42, a target molecule of integrin β1, and p110β expression. Knockdown of anyone of integrin β1, Cdc42 and p110β inhibited the activity of PI3K signaling, and sensitized DR-AsPC-1 cells to gemcitabine. GST pull-down assay showed that GTP-Cdc42 interacted with p110β. Collectively, these data indicated that integrin β1 promoted gemcitabine resistance in PDAC through Cdc42 activation of PI3K p110β signaling. In vivo experiments also confirmed this conclusion. These findings contribute to a better understanding the molecular mechanism of chemoresistance and facilitate the development of more targeted and effective treatment strategy for PDAC.

Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma

AIMTo clarify the underlying mechanism of formin-like 3 (FMNL3) in the promotion of colorectal carcinoma (CRC) cell invasion.METHODSThe in vitro biological function analyses of FMNL3 were performed by gain- and loss-of function approaches. Changes in the F-actin cytoskeleton were detected by the technologies of phalloidin-TRITC labeling and confocal microscopy. The signaling pathway mediated by FMNL3 was explored by western blot, gelatin zymograph assay, co-immunoprecipitation (co-IP), immunofluorescence co-localization, and glutathione S-transferase (GST) pull-down assay.RESULTSThe in vitro experimental results showed that FMNL3 significantly promoted the proliferation, invasion, and migration of CRC cells (P < 0.05 and P < 0.01). Moreover, FMNL3 regulated the remodeling of actin-based protrusions such as filopodia and lamellipodia in a RhoC-dependent manner. The western blot and gelatin zymograph assay results indicated that FMNL3 was involved in the RhoC/ focal adhesion kinase (FAK) pathway and acted as an effector of RhoC to activate the downstream signaling of p-FAK as well as p-MAPK and p-AKT. This resulted in the increased expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), and the subsequent promotion of CRC cell invasion. The results of TAE226, U0126 or Ly294002 treatment confirmed an essential role of FMNL3 in activation of the RhoC/FAK pathway and the subsequent promotion of CRC invasion. Co-IP, co-localization and GST pull-down assays showed the direct interaction of FMNL3 with RhoC in vivo and in vitro.CONCLUSIONFMNL3 regulates the RhoC/FAK signaling pathway and RhoC-dependent remodeling of actin-based protrusions to promote CRC invasion.

GtfA Interacting with GtfB is Required for PsrP Glycosylation in Streptococcus pneumoniae

Background: Streptococcus pneumoniae can adhere to lung cells via many adhesins, including pneumococcal serine-rich repeat protein (PsrP). Glycosyltransferase A (GtfA) and glycoslytransferase B (GtfB) were found to initiate PsrP glycosylation in Streptococcus pneumoniae serotype 4, isolate TIGR4. Objectives: To test whether GtfA and GtfB are conserved among S. pneumoniae isolates presenting with different serotypes and are involved in the glycosylation of PsrP via formation of the glycosyltransferase complex GtfA-GtfB. Methods: Serotyping of S. pneumoniae was performed using polymerase chain reaction (PCR) assays. The evolutionary divergences of GtfA and GtfB among different isolates were estimated using MEGA4 software. The contribution of GtfA and GtfB to the glycosylation of PsrP was identified using an in vivo glycosylation system in Escherichia coli. Interaction of GtfA with GtfB was assessed using a glutathione S-transferase (GST) pull-down assay and yeast two-hybrid experiment. Results: Nine different S. pneumoniae serotypes were found, of which the most frequent was 19F. The pneumococcal isolate TIGR4 and 22 other clinical isolates were included to estimate evolutionary divergence. The average homologies of the GtfA and GtfB sequences were 99.59% and 98.53%, respectively. Glycosyltransferase A and GtfB were shown to be required for the transfer of N-acetylglucosamine moieties to PsrP1-374. Glutathione S-transferase pull-down assays and yeast two-hybrid experiments showed that GtfA directly interacted with GtfB. Conclusions: Glycosyltransferase A and GtfB are conserved in S. pneumoniae isolates. Glycosyltransferase A interacts with GtfB, forming the glycosyltransferase complex GtfA-GtfB, which is required for PsrP glycosylation.

BCL3 regulates RANKL-induced osteoclastogenesis by interacting with TRAF6 in bone marrow-derived macrophages.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential component of the signaling complex that mediates osteoclastogenesis. As an adaptor protein of E3 ligase function, TRAF6 regulates NF-κB signaling via TAK1 and I-κB kinase (IKK) activation. Here, we investigated novel mechanisms by which TRAF6 signaling is regulated under receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. A yeast two-hybrid screen system identified cellular factors that interact with TRAF6. The interactions were confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays, followed by immuno-blotting. The role of TRAF6 in bone growth and remodeling was determined by osteoclast differentiation and bone-resorption pit assays. Regulatory mechanisms were examined by co-immunoprecipitation, immuno-blotting, real-time polymerase chain reaction, and luciferase reporter assays. We show that B-cell chronic lymphatic leukemia protein 3 (BCL3) interacts with TRAF6 through its ankyrin-repeat domain and inhibits osteoclastogenesis in bone marrow derived macrophages (BMDMs). Further, TRAF6 interacts with CYLD to mediate BCL3 deubiquitination, which facilitates the cytoplasmic accumulation of BCL3 and represses BCL3 and p50 complex-mediated cyclin D1 transcription. TRAF6 promotes RANKL-induced osteoclastogenesis by regulating novel non-canonical NF-κB signaling via BCL3 deubiquitination, indicating that BCL3 provides valuable insights into bone loss-associated diseases.

Tumor Necrosis Factor Receptor-Associated Factor 5 Interacts with the NS3 Protein and Promotes Classical Swine Fever Virus Replication.

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S-transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

FHC, an NS4B-interacting Protein, Enhances Classical Swine Fever Virus Propagation and Acts Positively in Viral Anti-apoptosis

Classical swine fever virus (CSFV), the etiological agent of classical swine fever, causes enormous economic loss to the pig industry. Ferritin heavy chain (FHC) is a notable anti-apoptotic protein, and existing evidence suggests that CSFV cannot induce apoptosis of host cells, however, the role of FHC in CSFV replication remains unclear. In the present study, we found that recombinant lentivirus-mediated knockdown or overexpression of FHC inhibited or enhanced CSFV replication, respectively, indicating a positive role for FHC in CSFV proliferation. Furthermore, interaction between the CSFV NS4B protein and FHC was confirmed by glutathione S-transferase (GST) pull-down, co-immunoprecipitation (co-IP) and confocal imaging assays. In addition, both CSFV replication and NS4B expression upregulated expression of FHC, which counteracts apoptosis by modulating cellular reactive oxygen species (ROS). These results suggest that FHC, an NS4B-interacting protein, enhances CSFV replication and has a positive role in viral anti-apoptosis by regulating ROS accumulation. This work may provide a new perspective for understanding the mechanism of CSFV pathogenesis.

An N-terminal motif unique to primate tau enables differential protein–protein interactions

Compared with other mammalian species, humans are particularly susceptible to tau-mediated neurodegenerative disorders. Differential interactions of the tau protein with other proteins are critical for mediating tau's physiological functions as well as tau-associated pathological processes. Primate tau harbors an 11-amino acid-long motif in its N-terminal region (residues 18-28), which is not present in non-primate species and whose function is unknown. Here, we used deletion mutagenesis to remove this sequence region from the longest human tau isoform, followed by glutathione S-transferase (GST) pulldown assays paired with isobaric tags for relative and absolute quantitation (iTRAQ) multiplex labeling, a quantitative method to measure protein abundance by mass spectrometry. Using this method, we found that the primate-specific N-terminal tau motif differentially mediates interactions with neuronal proteins. Among these binding partners are proteins involved in synaptic transmission (synapsin-1 and synaptotagmin-1) and signaling proteins of the 14-3-3 family. Furthermore, we identified an interaction of tau with a member of the annexin family (annexin A5) that was linked to the 11-residue motif. These results suggest that primate Tau has evolved specific residues that differentially regulate protein-protein interactions compared with tau proteins from other non-primate mammalian species. Our findings provide in vitro insights into tau's interactions with other proteins that may be relevant to human disease.

The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins

Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein–protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.

A Direct Interaction Between P53-Binding Protein 1 and Minichromosome Maintenance Complex in Hepg2 Cells

Background/Aims: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. DNA damage repair in cancer cells is a promising approach for the treatment of cancers. We aimed to explore the potential interaction between p53-binding protein 1 (53BP1) and minichromosome maintenance (MCMs) proteins during DNA damage in human hepatoma HepG2 cells. Methods: The recombinant vectors of 53BP1 and MCMs with tags were constructed and transfected into HepG2 cells. Immunoprecipitation (IP) and mass spectrometry (MS) were performed to identify the possible interactions between 53BP1 and MCMs, and glutathione S-transferase (GST) pull-down assay was carried out to detect the direct interaction. Moreover, the expressions of MCM2 and MCM6 were suppressed by specific short hairpin RNAs (shRNAs), and then the chromatin fraction and foci formation of 53BP1 were examined under the condition of DNA damage. Results: The results showed that MCM2/3/5/6 was immunoprecipitated against the hemaglutinin (HA)-tagged 53BP1 in HepG2 cell nuclei. GST results revealed that there was a direct interaction between 53BP1 and MCMs complex. Moreover, the non-chromatin level of 53BP1 was significantly increased by down-regulation of MCM2 or MCM6, but was statistically decreased the chromatin level. Furthermore, we observed that knockdown of MCM2 or MCM6 could statistically inhibit the foci formation of 53BP1 in HepG2 cell nuclei upon bleomycin-induced DNA damage (P < 0.01). Conclusion: Our results suggest that there is a direct interaction between 53BP1 and MCMs, which is essential for 53BP1 chromatin fraction and foci formation in hepatoma HepG2 cells.

The distinctive cell division interactome of Neisseria gonorrhoeae

BackgroundBacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW.ResultsUsing a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsANg were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW.ConclusionsResults from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.

Androgen enhances the activity of ETS-1 and promotes the proliferation of HCC cells.

The expression of androgen receptor (AR) has been detected in hepatocellular cancer (HCC). However, there is no universal model detailing AR’s function and mechanism in HCC. This study’s results show that treatment with dihydrotestosterone (DHT), an endogenous androgen, promoted HCC cells’ proliferation and up-regulated the transcription factor activity of ETS-1 (E26 transformation specific sequence 1), which mediates the migration and invasion of cancer cells via protein-protein interaction between AR and ETS-1. Results from luciferase assays showed that ETS-1’s activity was significantly up-regulated following androgen treatment. AR mediated ETS-1’s DHT-induced transcription factor activity. A potential protein-protein interaction between ETS-1 and AR was identified via glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. The mechanisms’ data indicated that enhancing AR activity increases ETS-1’s activity by modulating its cytoplasmic/nuclear translocation and recruiting ETS-1 to its target genes’ promoter. Moreover, while overexpression of AR significantly increased the proliferation or in vitro migration or invasion of HepG2 cells in the presence of androgen, inhibiting AR’s activity reduced these abilities. Thus, AR’s function as a novel ETS-1 co-activator or potentially therapeutic target of HCC has been demonstrated.