In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution.

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3'-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317-352-Hit1p70-164 complex reveals a novel mode of protein-protein association explaining the strong stability of the Rsa1p-Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p-Rsa1p-Hit1p heterotrimer.

Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.

Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression.

The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.

Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma.

TRIM24 (TIF1α), TRIM28 (TIF1ß), and TRIM33 (TIF1γ) are three related cofactors belonging to the tripartite motif superfamily that interact with distinct transcription factors. TRIM24 interacts with the liganded retinoic acid (RA) receptor to repress its transcriptional activity. Germ line inactivation of TRIM24 in mice deregulates RA-signaling in hepatocytes leading to the development of hepatocellular carcinoma (HCC). Here we show that TRIM24 can be purified as at least two macromolecular complexes comprising either TRIM33 or TRIM33 and TRIM28. Somatic hepatocyte-specific inactivation of TRIM24, TRIM28, or TRIM33 all promote HCC in a cell-autonomous manner in mice. Moreover, HCC formation upon TRIM24 inactivation is strongly potentiated by further loss of TRIM33. These results demonstrate that the TIF1-related subfamily of TRIM proteins interact both physically and functionally to modulate HCC formation in mice.

2-D structure of the A region of Xist RNA and its implication for PRC2 association.

In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger.

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP ß-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na(+)/Ca(2+) exchanger from squid nerve.

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions.

BACKGROUND: Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. RESULTS: Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. CONCLUSION: This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.

Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry.

This Feature will introduce the strategies of therapeutic antibodies (mAbs) in-depth characterization by mass spectrometry (MS) and discuss analytical comparison of biosimilar to originator mAbs, with the cases of trastuzumab and cetuximab. In addition, the structural and functional insights gained both by state-of-the art and emerging MS methods used for biobetters and next generation antibodies design and optimization will also be highlighted.

Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry.

Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex.

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.

The Pih1-Tah1 cochaperone complex inhibits Hsp90 molecular chaperone ATPase activity.

Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.

Combination of noncovalent mass spectrometry and traveling wave ion mobility spectrometry reveals sugar-induced conformational changes of central glycolytic genes repressor/DNA complex.

The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.

Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches.

The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1.

Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.

The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha.

Recent advances reveal emerging unique functions of poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 in heterochromatin integrity and cell differentiation. However, the chromatin-mediated molecular and cellular events involved remain elusive. Here we describe specific physical and functional interactions of Parp-1 and Parp-2 with the transcriptional intermediary factor (TIF1beta) and the heterochromatin proteins (HP1) that affect endodermal differentiation. We show that Parp-2 binds to TIF1beta with high affinity both directly and through HP1alpha. Both partners colocalize at pericentric heterochromatin in primitive endoderm-like cells. Parp-2 also binds to HP1beta but not to HP1gamma. In contrast Parp-1 binds weakly to TIF1beta and HP1beta only. Both Parps selectively poly(ADP-ribosyl)ate HP1alpha. Using shRNA approaches, we provide evidence for distinct participation of both Parps in endodermal differentiation. Whereas Parp-2 and its activity are required for the relocation of TIF1beta to heterochromatic foci during primitive endodermal differentiation, Parp-1 and its activity modulate TIF1beta-HP1alpha association with consequences on parietal endodermal differentiation. Both Parps control TIF1beta transcriptional activity. In addition, this work identifies both Parps as new modulators of the HP1-mediated subcode histone.-Quénet, D., Gasser, V., Fouillen, L., Cammas, F., Sanglier-Cianferani, S., Losson, R., Dantzer, F. The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha.

Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry.

BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs. MATERIAL/METHODS: Noncovalent electrospray ionization mass spectrometry (ESI-MS) was used to investigate bovine and human PEBP binding properties towards morphine and morphine-glucuronides. RESULTS: We describe for the first time that: (i) PEBP directly interacts with morphine glucuronides (M3G and M6G) but not with morphine, (ii) that the presence of a glucuronide group either on the 3rd or the 6th morphine's carbon does not affect these interactions, (iii) that M6G binds PEBP in a similar manner as the reference ligand PE and (iv) that PEBP displays a similar affinity for PE, M6G and M3G. CONCLUSIONS: Our results suggest that PEBP might protect M6G following its secretion into blood, leading to a longer half life. This study highlights the potentialities of ESI-MS to validate / invalidate the formation of protein: ligand noncovalent complexes when low affinity binders (i.e., compounds with affinities lower than 10(3) M(-1)) are concerned.