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ABSTRACT: Influenza A virus is an important pathogenic virus known to induce host cell cycle arrest in G0/G1 phase and create beneficial conditions for viral replication. However, how the virus achieves arrest remains unclear. We investigated the mechanisms underlying this process and found that the non-structural protein 1 (NS1) is required. Based on this finding, we generated a viable influenza A virus (H1N1) lacking the entire NS1 gene to study the function of this protein in cell cycle regulation. Besides some cell cycle regulators were changed, the concentration and activity of RhoA protein, which is thought to be pivotal for G1/S phase transition, was also decreased with over-expressing NS1. At the same time, cell cycle regulator pRb, the downstream of RhoA kinase's phosphorylation level was decreased in an NS1-dependent manner. These findings indicate that the NS1 protein induces G0/G1 cell cycle arrest mainly through interfering with the RhoA/pRb signalling cascade, thus providing favorable conditions for viral protein accumulation and replication. We further investigated the NS1 protein of avian influenza virus (H5N1) and found it also can decrease the expression and activity of RhoA, suggesting that the H5N1 virus may affect the cell cycle through the same mechanism. The NS1/RhoA/pRb cascade which can induce the G0/G1 cell cycle arrest identified here provides a unified explanation for the seemingly different NS1 functions involved in viral replication events. Our findings shed light on the mechanism of influenza virus replication and open new avenues for understanding the interaction between pathogens and hosts.
Journal of Virology 01/2013; · 5.40 Impact Factor
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ABSTRACT: The IKK/NF-κB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus. This pathway is therefore a prime target for viruses attempting to counteract the host response to infection. Here, we report that the influenza A virus NS1 protein specifically inhibits IKK-mediated NF-κB activation and production of the NF-κB induced antiviral genes by physically interacting with IKK through the C-terminal effector domain. The interaction between NS1 and IKKα/IKKβ affects their phosphorylation function in both the cytoplasm and nucleus. In the cytoplasm, NS1 not only blocks IKKβ-mediated phosphorylation and degradation of IκBα in the classical pathway but also suppresses IKKα-mediated processing of p100 to p52 in the alternative pathway, which leads to the inhibition of nuclear translocation of NF-κB and the subsequent expression of downstream NF-κB target genes. In the nucleus, NS1 impairs IKK-mediated phosphorylation of histone H3 Ser 10 that is critical to induce rapid expression of NF-κB target genes. These results reveal a new mechanism by which influenza A virus NS1 protein counteracts host NF-κB-mediated antiviral response through the disruption of IKK function. In this way, NS1 diminishes antiviral responses to infection and, in turn, enhances viral pathogenesis.
Cellular Microbiology 08/2012; · 5.46 Impact Factor
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Zhengguang Guo,
Eli Song,
Sucan Ma,
Xiaorong Wang, Shijuan Gao,
Chen Shao,
Siqi Hu,
Lulu Jia,
Rui Tian,
Tao Xu,
Youhe Gao
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ABSTRACT: Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.
Journal of Proteome Research 08/2012; 11(10):4847-62. · 5.11 Impact Factor
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ABSTRACT: This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.
Biomacromolecules 12/2011; 13(1):173-9. · 5.48 Impact Factor
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ABSTRACT: New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 05/2011; 27(5):773-80.
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ABSTRACT: RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors.
Protein & Cell 04/2011; 2(4):273-81.
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ABSTRACT: MicroRNAs (miRNAs) are a class of noncoding RNAs of lengths ranging from 18 to 23 nucleotides (nt) that play critical roles in a wide variety of biological processes. There is a growing amount of evidence that miRNAs play critical roles in intricate host-pathogen interaction networks, but the involvement of miRNAs during influenza viral infection is unknown. To determine whether the cellular miRNAs play an important role in H1N1 influenza A viral infections, 3' untranslated region (UTR) reporter analysis was used to identify putative miRNA targets in the influenza virus genome, and virus proliferation analysis was used to detect the effect of the screened miRNAs on the replication of H1N1 influenza A virus (A/WSN/33) in MDCK cells. The results showed that miRNA 323 (miR-323), miR-491, and miR-654 inhibit replication of the H1N1 influenza A virus through binding to the PB1 gene. Moreover mutational analysis of the predicted miRNA binding sites showed that the three miRNAs bind to the same conserved region of the PB1 gene. Intriguingly, despite the fact that the miRNAs and PB1 mRNA binding sequences are not a perfect match, the miRNAs downregulate PB1 expression through mRNA degradation instead of translation repression. This is the first demonstration that cellular miRNAs regulate influenza viral replication by degradation of the viral gene. Our findings support the notion that any miRNA has antiviral potential, independent of its cellular function, and that the cellular miRNAs play an important role in the host, defending against virus infection.
Journal of Virology 09/2010; 84(17):8849-60. · 5.40 Impact Factor
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ABSTRACT: Targeted gene delivery systems have attracted great attention due to their potential in directing the therapeutic genes to the target cells. However, due to their low efficiency, most of the successful applications of polymeric vectors have been focused on genes which can achieve robust expression. Minicircle DNA (mcDNA) is a powerful candidate in terms of improving gene expression and prolonging the lifespan of gene expression. In this study, we have combined folate/poly(ethylene glycol) modified polyethylenimine and mcDNA as a new tumor gene delivery system. We found that folate-labeled polyplexes were homogenous, with a size ranging from 60 to 85 nm. mcDNA increased folate-labeled vector based gene expression 2-8 fold in folate receptor-positive cells. Results of folic acid competition assay indicated that mcDNA mediated by folate-labeled vector were internalized into cells through receptor-mediated endocytosis. The investigation of the endocytosis pathway of the polyplexes showed that a large portion of them escaped from endo/lysosome and the polyplexes were associated before being separated in the nucleus. Furthermore, in vivo optical imaging and luciferase assays demonstrated that systemic delivery of the folate-labeled polyplexes resulted in preferential accumulation of transgenes in folate receptor-positive tumors, and mcDNA mediated approach achieved 2.3 fold higher gene expressions in tumors than conventional plasmid. Cytotoxicity assays showed that PEG-shielding of the polyplexes reduced the toxicity of PEI.
Biomaterials 08/2010; 31(23):6075-86. · 7.40 Impact Factor
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ABSTRACT: Polyethylenimine (PEI) is one of the most characterized non-viral vectors. It can condense DNA in a good manner and achieve high transfection efficiency. Minicircle DNA (mc-DNA) is a novel kind of supercoiled DNA which is devoid of bacterial backbone. mc-DNA is superior to conventional DNA for its higher transfection efficiency and longer time-span. In this study, we combined PEI and mc-DNA in gene delivery system. We investigated the physicochemical and biochemical effects of this non-viral system and further explore its potential in tumor gene therapy. mc-DNA was obtained by recombination of parental plasmid in the presence of L-arabinose, and complexed with PEI. The results of transmission electron microscopy and scanning electron microscopy showed that the particles were spherical and homogeneous. Through gel retardation assay and MTT assay, we found that there were no obvious differences in binding capability of PEI to mc-DNA and plasmid DNA, as well as in cytotoxicity. The results of dynamic light scattering showed that the size of PEI/mc-DNA was about 68 nm, a slight larger than that of PEI/plasmid DNA. Furthermore, the tumor cells transfected with mc-GFP showed higher GFP expression level than that of conventional plasmid. The same results were achieved in the cells treated with tumor-suppressor gene pten, assayed by RT-PCR and Western blot. It indicates that the system of PEI/minicircle DNA is promising in gene transfer.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2010; 26(6):772-9.
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ABSTRACT: PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two-hybrid screening of a random peptide library and high-throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three-hybrid system. Many G-protein signaling pathway-related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide-binding domains.
Cellular Physiology and Biochemistry 02/2009; 24(3-4):231-42. · 2.86 Impact Factor
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ABSTRACT: HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P(-3), which means that the P(-3) residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.
Science in China Series C Life Sciences 07/2007; 50(3):412-22. · 1.61 Impact Factor
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ABSTRACT: A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.
Molecular & Cellular Proteomics 09/2006; 5(8):1368-81. · 7.40 Impact Factor
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ABSTRACT: The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.
Molecular & Cellular Proteomics 05/2006; 5(4):769-76. · 7.40 Impact Factor
