[Screening of host cell proteins that interact with Toxoplasma gondii ROP18 via yeast two-hybrid system].

OBJECTIVE: To screen the host cell proteins that can interact with Toxoplasma gondii ROP18 by using yeast two-hybrid system. METHODS: The ROP18 gene fragments were amplified by RT-PCR from mRNA of T. gondii RH strain. The product of RT-PCR was digested with double restriction enzyme and was subcloned into the bait vector pGBKT7. The recombinant plasmid was transferred into yeast AH109 strain. Its toxicity and the autonomous activating activity were tested. The human fetal brain cDNA library was screened with pGBKT7-ROP18(25-251aa) as the bait plasmid by yeast two-hybrid system. RESULTS: The bait was constructed and AH109/PGBKT7-ROP18 showed an autonomous activity. The yeast strain AH109/pGBKT7-ROP18(25-251aa) line was then mated with the Mate & PlateTM Human Fetal Brain cDNA library. Using the selection procedures, eight novel host cell proteins were obtained: damage-specific DNA binding protein 1 (DDB1), torsin A interacting protein 1 (TOR1AIP1), integrin beta 1, solute carrier family 3 (SLC3A2), tyrosyl protein sulfotransferase (TPST2), OCIA domain containing 1 (OCIAD1), Derl-like domain family member 2 (DERL2), in addition to Homo sapiens activating transcription factor 6 beta(ATF6). CONCLUSION: Eight novel host cell proteins have been obtained via yeast two-hybrid system, which can interact with TgROP18.

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin ß1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins.

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.

Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag.

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.