Influenza A virus enhances its propagation through the modulation of Annexin-A1 dependent endosomal trafficking and apoptosis.

The influenza virus infects millions of people each year and can result in severe complications. Understanding virus recognition and host responses to influenza infection will enable future development of more effective anti-viral therapies. Previous research has revealed diverse yet important roles for the annexin family of proteins in modulating the course of influenza A virus (IAV) infection. However, the role of Annexin-A1 (ANXA1) in IAV infection has not been addressed. Here, we show that ANXA1 deficient mice exhibit a survival advantage, and lower viral titers after infection. This was accompanied with enhanced inflammatory cell infiltration during IAV infection. ANXA1 expression is increased during influenza infection clinically, in vivo and in vitro. The presence of ANXA1 enhances viral replication, influences virus binding, and enhances endosomal trafficking of the virus to the nucleus. ANXA1 colocalizes with early and late endosomes near the nucleus, and enhances nuclear accumulation of viral nucleoprotein. In addition, ANXA1 enhances IAV-mediated apoptosis. Overall, our study demonstrates that ANXA1 plays an important role in influenza virus replication and propagation through various mechanisms and that we predict that the regulation of ANXA1 expression during IAV infection may be a viral strategy to enhance its infectivity.

Annexin-1-deficient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen-specific antibody responses in a mouse model of asthma.

BACKGROUND: Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin-1 (ANXA1) is an anti-inflammatory protein which has been described as an endogenous protein responsible for some anti-inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1-deficient ((-/-)) mice are Th2 biased, and ANXA1 N-terminus peptide exhibits anti-inflammatory activity in a rat model of pulmonary inflammation. OBJECTIVE: ANXA1 protein is found in bronchoalveolar lavage fluid from asthmatics. However, the function of ANXA1 in the pathological development of allergy or asthma is unclear. Thus, in this study we intended to examine the effect of ANXA1 deficiency on allergen-specific antibody responses and airway responses to methacholine (Mch). METHODS: ANXA1(-/-) mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. Airway resistance, lung compliance and enhanced pause (PenH) were measured in naïve, sensitized and saline or allergen-challenged wild-type (WT) and ANXA1(-/-) mice. Total and allergen-specific antibodies were measured in the serum. RESULTS: We show that allergen-specific and total IgE, IgG2a and IgG2b levels were significantly higher in ANXA1(-/-) mice. Furthermore, naïve ANXA1(-/-) mice displayed higher airway hypersensitivity to inhaled Mch, and significant differences were also observed in allergen-sensitized and allergen-challenged ANXA1(-/-) mice compared with WT mice. CONCLUSIONS: In conclusion, ANXA1(-/-) mice possess multiple features characteristic to allergic asthma, such as airway hyperresponsiveness and enhanced antibody responses, suggesting that ANXA1 plays a critical regulatory role in the development of asthma. CLINICAL RELEVANCE: We postulate that ANXA1 is an important regulatory factor in the development of allergic disease and dysregulation of its expression can lead to pathological changes which may affect disease progression.

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis.

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKß. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

Nutlin-3 inhibits the NFkappaB pathway in a p53-dependent manner: implications in lung cancer therapy.

Nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53-Mdm2 interaction. We show in this study that Nutlin-3 can downregulate TNFalpha induced activation of the NFkappaB reporter in lung cancer cells. Activation of p53 dependent transcription is not compromised when Nutlin-3 is combined with TNFalpha. Instead, this combination treatment decreases cell viability in a p53 dependent manner. We show that Nutlin-3 strikingly inhibits the protein expression of NFkappaB target genes ICAM-1 and MCP-1 while other targets like Bcl-xL and FLIP are not affected, thereby suggesting that the inhibition is promoter specific. This inhibition of ICAM-1 and MCP-1 by Nutlin-3 is again dependent on the p53 status in cells. Furthermore, we show that Nutlin-3 strongly inhibits protein expression of ICAM-1 and MCP-1 induced by IL1, another NFkappaB activating stimuli. Nutlin-3 does not inhibit Akt phosphorylation, IkappaB alpha phosphorylation, IkappaB alpha degradation, p65 modification or p65 DNA binding in the cell lines tested. This study suggests the potential of Nutlin-3 as a bitargeted anti-cancer drug by simultaneously causing p53 activation and NFkappaB suppression. It also suggests that Nutlin-3 could be evaluated for treatment of lung cancer as a single agent or in combination therapy by targeting its effect on ICAM-1 and MCP-1 which are known to be critical for cancer cell invasion, thereby downregulating tumor formation and metastasis. This study also suggests biomarkers of response for evaluation of Nutlin-3 in the clinic.

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.

E. coli expressed proteins as diagnostic reagents for typing of foot-and-mouth disease virus.

Truncated proteins corresponding to the C-terminal half of VP1 of four vaccine strains and two field variants of foot-and-mouth disease virus (FMDV) were expressed in E. coli. The expressed proteins were affinity purified and their type specific reactivity was confirmed by immunoprecipitation with anti-virus antibodies. Antibodies were raised against the purified proteins in guinea pigs and the type specificity of the anti peptide antibodies was confirmed by antigen capture reverse transcription polymerase chain reaction (Ag-RT/PCR) where the sera against a particular type captured the homologous virus. Antibodies were purified by immuno-affinity chromatography and tested for specificity by various serological tests. Using the purified proteins and the antibodies raised against them, tests like ELISA, Ag-RT/PCR, and latex agglutination test (LAT) were standardized. Application of the reagents in various tests was studied by screening a few field samples and by nucleotide sequencing. Specific reactivity of antibodies raised against expressed protein was seen with both vaccine virus and field samples. Thus E. coli expressed proteins and antibodies to them may form an alternative and cheap source of diagnostic reagents. The studies showed that antibodies against peptides were mono-specific and therefore may be used in LAT for rapid typing of FMDV and Ag-RT/PCR for typing ELISA negative field samples.

Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.

Serotyping of foot-and-mouth disease virus by antigen capture reverse transcriptase/polymerase chain reaction.

The technique of capturing of foot-and-mouth disease virus (FMDV) from clinical material in microcentrifuge tubes coated with type-specific antibodies and amplifying the viral sequences by RT/PCR in the same tube, promoted the detection and serotyping of FMDV with high sensitivity and specificity. The efficiency of antigen capturing and shelf life of the coated tubes was improved by glutaraldehyde fixation of antibodies to the tubes. Virus in infected tissues, even after storage for 25-30 years at 70 degrees C, could be successfully typed by this method. Conserved sequences flanking the variable region of immunoreactive VP1 gene of FMDV were used as primers in the assay and hence the nucleotide sequence analysis of the product could reveal the strain variation. The test has been found to be at least 125-fold more sensitive than type specific ELISA and of comparable sensitivity as other protocols for detection of FMDV by RT/PCR.