Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein.

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.     

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor‐type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPρ is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto‐domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto‐domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane–proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain‐specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.     

A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells

DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185^neu expressing tumor MBT-2 in mice. We found that recombinant LZ-8 stimulated mouse bone marrow-derived dendritic cells (DCs) via TLR4 and its stimulatory effect was not due to any microbe contaminant. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, LZ-8 cotreatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase in antitumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.     

A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.     

Eps8 protein facilitates phagocytosis by increasing TLR4-MyD88 protein interaction in lipopolysaccharide-stimulated macrophages

Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97(Eps8)) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.     

细胞粘附分子群相互作用网络模型研究

通过相互作用网络可视化软件Cytoscape对整联蛋白粘合体156种成份及相互间的690个反应实现网络可视化,并通过网络分析软件network analyser得到该网络节点间的平均连接并不复杂是一个不可分割的整体网络,网络较稳定,信息传递很快等。直接可视化的网络通过节点的形状改变视图有助于我们在原始数据的基础上更好的认识这一复杂的作用网络的相关信息。     

An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction

Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600 g and 35 °C for 60 min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.     

Lipopolysaccharide recognition protein, MD-2, facilitates cellular uptake of E. coli-derived plasmid DNA in synovium

BACKGROUND: Several cell types are susceptible to transfection in vivo using naked plasmid DNA. The mechanisms involved in mediating in vivo transfection are incompletely known, but evidence suggests that receptor-mediated endocytosis is important for specific types of cells. In this study we tested the hypothesis that residual Escherichia coli lipopolysaccharide (LPS) forms a non-covalent complex with expression plasmid DNA, and host-cell-derived soluble LPS-binding proteins bind to the DNA-LPS complexes in order to facilitate receptor-mediated endocytosis. METHODS: Cells from the murine synovial lining were used as an in vivo model system and in vivo luciferase imaging was used to quantify levels of transgene expression. Using a series of gene-deleted mice, the roles of LPS recognition complex proteins, lipopolysaccharide-binding protein (LBP), CD14 and MD-2, in the process of in vivo transfection were determined. RESULTS: Luciferase expression assays revealed that mice lacking LBP or CD14 had increased luciferase expression (p < 0.023 and < 0.165, respectively), while mice deleted of MD-2 had significant reductions in luciferase expression (p < 0.001). Gene deletion of hyaluronic acid binding protein CD44 was used as a control and had no statistically significant effect on transgene expression in vivo. In muscle tissue, where neither cell surface nor soluble MD-2 is expressed, no MD-2 dependence of plasmid transfection was identified, suggesting the role of MD-2 is tissue or cell type specific. Additionally, depleting mice of macrophages showed that luciferase expression is occurring within fibroblast-like synoviocytes. CONCLUSIONS: Our data support a physical association between LPS and E. coli-derived plasmid DNA, and that in vivo transfection of fibroblast-like synoviocytes is dependent on the soluble form of the LPS-binding protein MD-2.     

An unknown genetic defect increases venous thrombosis risk, through interaction with protein C deficiency.

We used two-locus segregation analysis to test whether an unknown genetic defect interacts with protein C deficiency to increase susceptibility to venous thromboembolic disease in a single large pedigree. Sixty-seven pedigree members carry a His107Pro mutation in the protein C gene, which reduces protein C levels to a mean of 46% of normal. Twenty-one carriers of the mutation and five other pedigree members had verified thromboembolic disease. We inferred the presence in this pedigree of a thrombosis-susceptibility gene interacting with protein C deficiency, by rejecting the hypothesis that the cases of thromboembolic disease resulted from protein C deficiency alone and by not rejecting Mendelian transmission of the interacting gene. When coinherited with protein C deficiency, the interacting gene conferred a probability of a thrombotic episode of approximately 79% for men and approximately 99% for women, before age 60 years.     

The limited immunomodulatory effects of escharectomy on the kinetics of endotoxin, cytokines, and adhesion molecules in major burns

Escharectomy has been shown to improve the survival rates and the outcomes in burns. This observational study was conducted to assess the role of escharectomy on the inflammatory mediators in major burns. Seventeen ASA physical status II or status III adult surviving major burn patients were recruited. When the escharectomy was scheduled, a series of blood samples was obtained at -3 and -1 days preoperation, and +1 and +3 postoperation. The changing levels of endotoxin, cytokines, and adhesion molecules were measured with a quantitative sandwich immunoassay. Extensive escharectomy did not appear to have any significant impact on the levels of tumor necrosis factor alpha, interleukin-10, soluble intracellular adhesion molecule-1 and soluble vascular adhesion molecule-1. Meanwhile, endotoxin and E-selectin were significantly decreased after escharectomy. Escharectomy appeared to have a limited immunomodulatory effect on the inflammatory mediators in systemic inflammatory responses induced by major burns. This is probably related to the timing and extent of surgery, and the complex nature of burn-related inflammation.     

Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase

In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.     

The role of cellular adhesion molecules in virus attachment and entry

As obligate intracellular parasites, viruses must traverse the host-cell plasma membrane to initiate infection. This presents a formidable barrier, which they have evolved diverse strategies to overcome. Common to all entry pathways, however, is a mechanism of specific attachment to cell-surface macromolecules or ‘receptors’. Receptor usage frequently defines viral tropism, and consequently, the evolutionary changes in receptor specificity can lead to emergence of new strains exhibiting altered pathogenicity or host range. Several classes of molecules are exploited as receptors by diverse groups of viruses, including, for example, sialic acid moieties and integrins. In particular, many cell-adhesion molecules that belong to the immunoglobulin-like superfamily of proteins (IgSF CAMs) have been identified as viral receptors. Structural analysis of the interactions between viruses and IgSF CAM receptors has not shown binding to specific features, implying that the Ig-like fold may not be key. Both proteinaceous and enveloped viruses exploit these proteins, however, suggesting convergent evolution of this trait. Their use is surprising given the usually occluded position of CAMs on the cell surface, such as at tight junctions. Nonetheless, the reason for their widespread involvement in virus entry most probably originates in their functional rather than structural characteristics.     

The modulation of cellular contractility and adhesion by trypsin and EGTA

The relationship of cell to substrate and the organisation of the cellular contractile system has been studied using whole-cell mount transmission electron microscopy (TEM). The cellular response to trypsin is compared with that induced by treatment with the Ca²⁺-specific chelating agent EGTA. Trypsinisation results in a breakdown of the ordered contractile system and both cell-cell and cell-substrate adhesion. Studies with the respiratory inhibitor 2,4 dinitrophenol (DNP) have shown that cellular rounding mediated by trypsinisation is energy independent. The response to EGTA is confluence modulated. Confluent monolayers are induced to contract and detach as sheets, a response related here to the overlapping of the peripheral cytoplasm. Subconfluent cells contract centripetally withdrawing organelles to the perinuclear region. Withdrawal of organelles is accompanied by a redistribution of the contractile system. Contraction results in a rounded cell anchored to the substrate by numerous retraction fibrils. The response to EGTA is energy dependent. The observations suggest that a chelation of Ca²⁺ results in an active contraction of the actomyosin system. We propose a mechanism for this response based on ultrastructural and biochemical characterisation of the actomyosin system.     

Investigation of immunomodulatory and anti-inflammatory effects of eriodictyol through its cellular anti-oxidant activity

Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses against infection or to ameliorate immune-based pathologies. To determine whether eriodictyol has immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we investigated the stimulatory effect of eriodictyol on spleen cells isolated from BALB/c mice. Eriodictyol significantly stimulated splenocyte proliferation. However, only B lymphocytes (not T lymphocytes) could be stimulated by eriodictyol in a dose-related manner. Studies assessing potential effect of eriodictyol on innate immunity reported that eriodictyol enhanced significantly the killing activity of natural killer (NK) cells, T lymphocytes, and macrophages. We also demonstrated that eriodictyol inhibited nitric oxide (NO) production and lysosomal enzyme activity in murine peritoneal macrophages cultured ex-vivo, suggesting a potential anti-inflammatory effect in situ. Eriodictyol revealed also a cellular anti-oxidant activity in splenocytes and macrophages. Furthermore, eriodictyol increased catalase activity in spleen cells. From this data, it can be concluded that eriodictyol exhibited an immunomodulatory effect that could be ascribed in part to a cytoprotective effect related to its anti-oxidant activity.     

An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.     

Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.     

Regulation of the rhodopsin protein phosphatase, RDGC, through interaction with calmodulin.

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. In the current work, we demonstrate that RDGC binds to calmodulin, and a mutation in an IQ motif that eliminates the calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. Our data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC.