衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

Dis3‐like 1: a novel exoribonuclease associated with the human exosome

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.     

Hypoxia enhances antibody‐dependent dengue virus infection

Dengue virus (DENV) has been found to replicate in lymphoid organs such as the lymph nodes, spleen, and liver in post‐mortem analysis. These organs are known to have low oxygen levels (~0.5–4.5% O₂) due to the vascular anatomy. However, how physiologically low levels of oxygen affect DENV infection via hypoxia‐induced changes in the immune response remains unknown. Here, we show that monocytes adapted to 3% O₂ show greater susceptibility to antibody‐dependent enhancement of DENV infection. Low oxygen level induces HIF1α‐dependent upregulation of fragment crystallizable gamma receptor IIA (FcγRIIA) as well as HIF1α‐independent alterations in membrane ether lipid concentrations. The increased FcγRIIA expression operates synergistically with altered membrane composition, possibly through increase membrane fluidity, to increase uptake of DENV immune complexes for enhanced infection. Our findings thus indicate that the increased viral burden associated with secondary DENV infection is antibody‐dependent but hypoxia‐induced and suggest a role for targeting hypoxia‐induced factors for anti‐dengue therapy.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

A novel anti‐apoptotic role for apolipoprotein L2 in IFN‐γ‐induced cytotoxicity in human bronchial epithelial cells

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN‐γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN‐γ‐stimulated normal human bronchial epithelial cells (HBEC) using a 2‐dimensional gel electrophoresis followed by MALDI‐TOF‐MS/MS. IFN‐γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN‐γ, in lung tissues from an IFN‐γ‐predominant influenza A virus‐infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN‐γ‐induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN‐γ‐induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub‐G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN‐γ‐induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide‐double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase‐dependent manner. Our results reveal a novel function for ApoL2 in conferring anti‐apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN‐γ, in maintaining airway epithelial layer integrity. J. Cell. Physiol. 226: 397–406, 2011. © 2010 Wiley‐Liss, Inc.     

MXRA5 is a TGF‐β1‐regulated human protein with anti‐inflammatory and anti‐fibrotic properties

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling‐associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor‐β1 (TGFβ1) up‐regulated MXRA5 mRNA and protein expression. TGFβ1‐induced MXRA5 up‐regulation was prevented by either interference with TGFβ1 activation of the TGFβ receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro‐inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA‐induced down‐regulation of constitutive MXRA5 expression resulted in higher TWEAK‐induced expression of chemokines. In addition, MXRA5 down‐regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFβ1. Furthermore, in clear cell renal cancer, von Hippel–Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFβ1‐ and VHL‐regulated protein and, for the first time, we identify MXRA5 functions as an anti‐inflammatory and anti‐fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.     

Evaluation of tubulin β‐3 as a novel senescence‐associated gene in melanocytic malignant transformation

Malignant melanoma might develop from melanocytic nevi in which the growth‐arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth‐arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β‐3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β‐3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β‐3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β‐3 as a candidate marker.     

Identification of ciliary neurotrophic factor receptor α as a mediator of neurotoxicity induced by α‐synuclein

Accumulating evidence suggests that extracellular α‐synuclein (eSNCA) plays an important role in the pathogenesis of Parkinson's disease or related synucleinopathies by inducing neurotoxicity directly or indirectly via microglial or astroglial activation. However, the mechanisms by which this occurs remain to be characterized. To explore these mechanisms, we combined three biochemical techniques – stable isotope labeling of amino acid in cell cultures (SILAC), biotin labeling of plasma membrane proteins followed by affinity purification, and analysis of unique proteins binding to SNCA peptides on membrane arrays. The SILAC proteomic analysis identified 457 proteins, of which, 245 or 172 proteins belonged to membrane or membrane associated proteins, depending on the various bioinformatics tools used for interpretation. In dopamine neuronal cells treated with eSNCA, the levels of 86 membrane proteins were increased and 35 were decreased compared with untreated cells. In peptide array analysis, 127 proteins were identified as possibly interacting with eSNCA. Of those, seven proteins were overlapped with the membrane proteins that displayed alterations in relative abundance after eSNCA treatment. One was ciliary neurotrophic factor receptor, which appeared to modulate eSNCA‐mediated neurotoxicity via mechanisms related to JAK1/STAT3 signaling but independent of eSNCA endocytosis.     

Beyond attachment: Roles of DC‐SIGN in dengue virus infection

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN), a C‐type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC‐SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand‐off to another receptor or a co‐receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild‐type DC‐SIGN or 2 internalization‐deficient DC‐SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super‐resolution imaging and high content single particle tracking, we investigated DENV binding, DC‐SIGN surface transport, endocytosis, as well as cell infectivity. DC‐SIGN was found colocalized with DENV inside cells suggesting hand‐off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC‐SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co‐receptor because cells expressing the internalization‐deficient mutants could still be infected.      

Conformational flexibility of σ70 in anti‐terminator loading

In promoter DNA, the preferred distance of the ?10 and ?35 elements for interacting with RNA polymerase‐bound σ⁷⁰ is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C‐terminal domain of σ⁷⁰, including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing σ⁷⁰.     

Fibroblast growth factor 2‐antagonist activity of a long‐pentraxin 3‐derived anti‐angiogenic pentapeptide

Fibroblast growth factor‐2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long‐pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2‐antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2‐binding region PTX3‐(97–110) were assessed for their FGF2‐binding capacity. Among them, the shortest pentapeptide Ac‐ARPCA‐NH₂ (PTX3‐[100–104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2‐dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac‐ARPCA‐NH₂ inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2‐overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine‐kinase FGF receptor‐1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac‐ARPS A‐NH₂ peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF‐binding domain of the Ig‐like loop D2 of FGFR1, amino acid substitutions in Ac‐ARPCA‐NH₂ and saturation transfer difference‐nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3‐derived anti‐angiogenic FGF2 antagonists.     

A novel function for proSAAS as an amyloid anti‐aggregant in Alzheimer's disease

Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti‐aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12‐month‐old APdE9 mice, and in the cortex of a human AD‐affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co‐immunoprecipitated with Aβ immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aβ_1–42 at molar ratios of 1 : 10, and this anti‐aggregation effect was dose dependent. Structure‐function studies showed that residues 97–180 were sufficient for the anti‐aggregation function against Aβ. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral‐mediated proSAAS over‐expression, blocked the neurocytotoxic effect of Aβ_1–42 in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimer's disease pathology.

     

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

Persicarin is anti‐inflammatory mediator against HMGB1‐induced inflammatory responses in HUVECs and in CLP‐induced sepsis mice

High mobility group box 1 (HMGB1) protein is a crucial nuclear cytokine that mediates inflammatory responses, whereas persicarin is an active compound from Oenanthe javanica that has been widely researched for its neuroprotective and antioxidant activities. However, little is known of the effects of persicarin on HMGB1‐mediated inflammatory response. Here, we investigated this issue by monitoring the effects of persicarin on the lipopolysaccharide (LPS) and on the cecal ligation and puncture (CLP)‐mediated releases of HMGB1 and the effects of persicarin on the HMGB1‐mediated modulation of inflammatory response. Persicarin potently inhibited the release of HMGB1 and down‐regulated HMGB1‐dependent inflammatory responses in human endothelial cells, and inhibited HMGB1‐mediated hyperpermeability and leukocyte migration in mice. Furthermore, persicarin reduced CLP‐induced HMGB1 release and sepsis‐related mortality. Given these results, persicarin should be viewed as a candidate therapeutic for the treatment of severe vascular inflammatory diseases, such as, sepsis or septic shock. J. Cell. Physiol. 228: 696–703, 2013. © 2012 Wiley Periodicals, Inc.     

Comparison of the three‐dimensional structures of a humanized and a chimeric Fab of an anti‐γ‐interferon antibody

The objective of this work is to compare the three‐dimensional structures of “humanized” and mouse–human chimeric forms of a murine monoclonal antibody elicited against human γ‐interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen‐binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8000 by nearly identical microseeding procedures. Their structures were solved by X‐ray analyses at 2.9 Å resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity‐determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized V_H, the substitution of serine for proline in position 7 allowed the N‐terminal segment (designated strand 4‐1) to be closely juxtaposed to an adjacent strand (4‐2) and form hydrogen bonds typical of an antiparallel β‐pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an α‐helix involving residues 25–32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 Å to a random coil which terminated in a single turn of 3₁₀ helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space‐saving α‐helix, contrasting with a more extended 3₁₀ helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use. Copyright © 1999 John Wiley & Sons, Ltd.     

Human TLR3 recognizes dengue virus and modulates viral replication in vitro

The elicitation of large amount inflammatory cytokine in serum has been developed as the cause of the plasma leakage in dengue fever (DF)/dengue haemorrhagic fever (DHF) infection. Virus recognition in innate immunity is the key. The Toll-like receptors (TLRs) play an important role in pathogen recognition towards cytokine induction among several viruses; however, the role of TLRs on innate immune recognition against DENV remains unclear. This study aims at the interaction between dengue virus (DENV) and human TLRs at the incipient stage of infection in vitro . Our experiment reveals that stably expression of TLR3, 7, 8 on HEK293 enables IL-8 secretion after DENV recognition. By the model of human monocytic cells U937, we demonstrated the trigger of IL-8 after viral recognition of human monocytic cell is primary through TLR3 following endosomal acidification. Silencing of TLR3 in U937 cells significantly blocks the DENV-induced IL-8 production. Besides, the interaction is further corroborated by colocalization of TLR3 and DENV RNA upon DENV internalization. Furthermore, in this study we found the expression of TLR3 can mediate strong IFN-α/β release and inhibit DENV viral replication significantly, thus limit the cytopathic effect.