Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

1H, 15N and 13C resonance assignments of CG7054, a new PEBP from Drosophila melanogaster

International audienceCG7054 is a 179 residue protein from Drosophila melanogaster. Based on sequence alignment, it has been shown that CG7054 belongs to the phosphatidylethanolamine binding proteins (PEBP) family. PEBP proteins are widely distributed in various species, from bacteria to mammals. PEBPs have an affinity for anionic ligands, such as phosphatidylethanolamine, opioids, nucleotides (Bucquoy et al. 1994). Human PEBP-1 is implicated in cancer, acting as a metastase suppressor. During cancer treatment, it sensibilises cells to chemotherapy and immunotherapy. Human PEBP-1 is also implicated in other diseases (Alzheimer's disease, infertility, diabetes...). However, the molecular mechanisms by which PEBPs act remain obscure. These proteins modulate important cell mechanisms involving serine proteases (thrombin, neuropsin and chymotrypsin), G-proteins, MAP-kinase and NFkB signaling pathways. Several crystallographic studies (Serre et al. 1998) have shown that PEBPs share a similar topology, but the solution structure is unknown. In order to gain new insights into structurefunction relationships, dynamics and protein-protein or protein-ligand interactions in solution for this family of proteins, we present herein the assignments (1H, 13C, 15N) of the backbone (complete) and side chain (94%) of CG7054 using conventional triple resonance experiments. The chemical shifts have been deposited in the BioMagResBank under Accession No. 7286

Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogaster

The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFκB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure–function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly β-sheets at pH 7. The good dispersion of the crosspeaks on the 1H–15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family

EXPRESSION AND CHARACTERIZATION OF THE PEBP HOMOLOG GENES FROM Drosophila

International audienceThe phosphatidylethanolamine binding proteins (PEBPs) family is evolutionarily conserved and involved in different physiological phenomena. PEBPs were found in many species from bacteria to mammals. Despite numerous studies, PEBPs' biological function and mode of action remain elusive. Based on sequence homology, seven PEBP genes were detected in the Drosophila genome. Only one of them, the odorant binding protein (OBP), has been characterized. To date nothing is known concerning the expression pattern and biological roles of the six other PEBP genes. By RT-PCR and Western blot analysis, we examined expression of the PEBPs in different tissues and embryos. The 6 PEBPs were differentially expressed. Only one, CG10298, is specific of only one tissue: the testis. Additionally, by comparing in wild type and male-sterile mutants we show that CG10298 is present only during spermatid differentiation. Furthermore, by comparing structural parameters of the six PEBP proteins with those of human PEBP-1, we have established that PEBP CG10298 is most closely related to human PEB

Legume AG41 peptide: a promising bio-insecticide

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