21 research outputs found
FFAT motif phosphorylation controls formation and lipid transfer function of interâorganelle contacts
Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAPâA, VAPâB, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAPâA, VAPâB, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a nonâconventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for nonâconventional FFAT motifs (named PhosphoâFFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and PhosphoâFFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a PhosphoâFFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ERâendosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for interâorganelle contacts
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
ProFeatMap: a customizable tool for 2D feature representation of protein sets
ABSTRACT Summary Here, we present ProFeatMap, an intuitive Python-based website allowing to quickly display protein features such as domains, repeats, post-translational modifications location and so forth, into a highly customizable graphical 2D map. Starting from a user-defined protein list, ProFeatMap automatically extracts the main protein features from the Uniprot database. The resulting high-quality maps can help to gain insights, e.g. feature redundancy, that were previously overlooked but which may be useful for the research project. ProFeatMap is freely accessible on the web at: https://profeatmap.pythonanywhere.com/ Availability Source code is freely accessible at https://github.com/profeatmap/ProFeatMap under the GPL license. Contact [email protected] , [email protected] Supplementary information detailed user guide of ProFeatMa
Domain substructure of HPV E6 oncoprotein: biophysical characterization of the E6 Câterminal DNAâbinding domain
E6 is a viral oncoprotein implicated in cervical cancers, produced by highârisk human papillomaviruses (HPVs). Structural data concerning this protein are scarce due to the difficulty of producing recombinant E6. Recently, we described the expression and purification of a stable, folded, and biologically active HPV16 E6 mutant called E6 6C/6S. Here, we analyzed the domain substructure of this mutated E6. Nonspecific proteolysis of fullâlength E6 6C/6S (158 residues) yielded Nâterminal and Câterminal fragments encompassing residues 7â83 and 87â158, respectively. The Câterminal fragment of residues 87â158 was cloned, overexpressed, and purified at concentrations as high as 1 mM. The purified domain retains the selective fourâway DNA junction recognition activity of the fullâlength E6 protein. Using UV absorption, UV fluorescence, circular dichroism, and nuclear magnetic resonance, we show that the peptide is primarily monomeric and folded with equal proportions of alphaâhelix and betaâsheet secondary structur
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 (ÏPEI), which interacts with proteins and promotes their delivery into the cytosol of mammalian
cells. In aqueous medium at pH 7.4, self-association of ÏPEI 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
ÏPEI-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