Protein Expr Purif

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration

Intracellular delivery of functionally active proteins using self-assembling pyridylthiourea-polyethylenimine

Intracellular delivery of functionally active proteins into cells is emerging as a novel strategy for research and therapeutic applications. Here, we present the properties of a self-assembling pyridylthiourea-modified polyethylenimine (πPEI), which interacts with proteins and promotes their delivery into the cytosol of mammalian cells. In aqueous medium at pH 7.4, self-association of πPEI in the presence of green fluorescent proteins (GFP) leads to supramolecular protein-entrapped assemblies. These assemblies protect GFP from losing its fluorescence upon pH variation and assist delivery/translocation into the cytosol of mammalian cells via the endocytic pathway. The scope of application of this delivery system was extended to antibodies against intracellular targets as illustrated using a monoclonal antibody directed against the HPV-16 viral E6 oncoprotein and an antibody directed against the threonine-927 phosporylation site of the EG5 kinesin spindle protein. The πPEI-mediated delivery of native anti-E6 antibodies or anti-E6 antibodies equipped with a nuclear localization signal (NLS), led to regeneration of the p53 tumor suppression protein in E6-transformed CaSki cells. Delivery of functionally active anti-EG5 antibodies, with the same polymer, reduced HeLa cell viability and appeared to perturb, as expected, chromosome segregation during mitosis. Altogether, these results provide an easy to use delivery system for extending the scope of application of antibodies for epitope recognition within living cells and may provide novel opportunities for selective interference of cell function by a steric hindrance modality

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

International audienceMany protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes