Enhanced stability designs of cell membrane chromatography for screening drug leads.

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.

Targeting and Covalently Immobilizing the EGFR through SNAP-Tag Technology for Screening Drug Leads.

Membrane protein immobilization is particularly significant in in vitro drug screening and determining drug-receptor interactions. However, there are still some problems in the immobilization of membrane proteins with controllable direction and high conformational stability, activity, and specificity. Cell membrane chromatography (CMC) retains the complete biological structure of membrane proteins. However, conventional CMC has the limitation of poor stability, which results in its limited life span and low reproducibility. To overcome this limitation, we propose a method for the specific covalent immobilization of membrane proteins in cell membranes. We used the SNAP-tag as an immobilization tag fused to the epidermal growth factor receptor (EGFR), and Cys145 located at the active site of the SNAP-tag reacted with the benzyl group of O6-benzylguanine (BG). The SNAP-tagged EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR from the cell membrane suspension onto a BG-derivative-modified silica gel. Our immobilization strategy improved the life span and specificity of CMC and minimized loss of activity and nonspecific attachment of proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system was established to screen EGFR antagonists from Epimedii folium. Icariin, magnoflorine, epimedin B, and epimedin C were retained in this model, and pharmacological assays revealed that magnoflorine could inhibit cancer cell growth by targeting the EGFR. This EGFR immobilization method may open up possibilities for the immobilization of other membrane proteins and has the potential to serve as a useful platform for screening receptor-binding leads from natural medicinal herbs.

Effect of yessotoxin on cytosolic calcium levels in human hepatocellular carcinoma cells in vitro.

Yessotoxin (YTX) and its analogs are a type of marine toxins found in marine environments in numerous coastal countries. These toxins tend to accumulate in filter-feeding molluscs and may threaten the shellfish industry and public health. Several previous studies indicated that YTX may induce apoptosis in different types of cell lines, although the exact underlying mechanisms have not yet been elucidated. The aim of this study was to mainly focus on the effect of YTX on cytosolic Ca2+ levels in human hepatocellular carcinoma cells. In order to investigate the exact mechanism of YTX-evoked Ca2+ increase, laser scanning confocal microscopy was used, with the addition of the chelator ethylene glycol tetraacetic acid (EGTA) and nifedipine, an L-type Ca2+ channel blocker, to the reaction system. The results demonstrated that YTX caused cytosolic Ca2+ level increase in Bel7402 cells and the YTX-evoked Ca2+ increase was successfully blocked by EGTA and nifedipine. Therefore, our results indicated that YTX may cause apoptosis via inducing Ca2+ entry in Bel7402 cells.

Characterization of apoptotic changes induced by yessotoxin in the Bel7402 human hepatoma cell line.

The apoptotic effects of yessotoxin (YTX) on Bel7402 human hepatoma cells have been evaluated by the combination of several methods, including optical microscopy, Hoechst 33342 staining and DNA gel electrophoresis. Rhodamine 123 staining has also been used to investigate changes in mitochondrial transmembrane potential. The results indicated that YTX has negative effects on human liver cells and induces apoptosis. Furthermore, changes in calcium concentrations were analyzed using the fluorescence probe Fluo-3 AM. The results showed that intracellular calcium concentrations increased after treatment with YTX.

Apoptosis induced by yessotoxins in Hela human cervical cancer cells in vitro.

To investigate the apoptotic effects of an extract of red-tide algae, Protoceratium reticulatum, on HeLa cells, we used liquid chromatography-tandem mass spectrometry, optical microscopy, Hoechst 33342/propidine iodide staining, and DNA gel electrophoresis to analyze its constituents and toxicity, as well as rhodamine 123 staining to investigate changes in mitochondrial membrane potential. Analysis showed that the P. reticulatum extract contained the yessotoxins (YTXs) homo-YTX, 45-OH-YTX and 45-OH-homo-YTX. The results indicated that P. reticulatum extract negatively influences HeLa cells and induces their apoptosis.

A Pyk2-Vav1 complex is recruited to beta3-adhesion sites to initiate Rho activation.

Integrin alphavbeta3-mediated adhesion of haemopoietic cells to vitronectin results in beta3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with alphavbeta3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alphavbeta3. The association of Pyk2 and Vav1 with beta3 relies on the presence of Tyr747 in beta3, the primary site of beta3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)-Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and beta3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery.

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

Purified integrin adhesion complexes exhibit actin-polymerization activity.

BACKGROUND: Cell adhesion and motility are accomplished through a functional linkage of the extracellular matrix with the actin cytoskeleton via adhesion complexes composed of integrin receptors and associated proteins. To determine whether this linkage is attained actively or passively, we isolated integrin complexes from nonadherent hematopoietic cells and determined their influence on the polymerization of actin. RESULTS: We observed that alpha(V)beta3 complexes are capable of dramatically accelerating the rate of actin assembly, resulting in actin fibers tethered at their growing ends by clustered integrins. The ability to enhance actin polymerization was dependent upon Arg-Gly-Asp-ligand-induced beta3 tyrosine phosphorylation, agonist-induced cellular activation, sequestration of Diaphanous formins, and clustering of the receptor. CONCLUSIONS: These results suggest that adhesion complexes actively promote actin assembly from their cytosolic face in order to establish a mechanical linkage with the extracellular matrix.

Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes.

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.