A new potential anti-cancer beta-carboline derivative decreases the expression levels of key proteins involved in glioma aggressiveness: A proteomic investigation.

Gliomas remain highly fatal due to their high resistance to current therapies. Deregulation of protein synthesis contributes to cancer onset and progression and is a source of rising interest for new drugs. CM16, a harmine derivative with predicted high blood-brain barrier penetration, exerts antiproliferative effects partly through translation inhibition. We evaluated herein how CM16 alters the proteome of glioma cells. The analysis of the gel-free LC/MS and auto-MS/MS data showed that CM16 induces time- and concentration-dependent significant changes in the total ion current chromatograms. In addition, we observed spontaneous clustering of the samples according to their treatment condition and their proper classification by unsupervised and supervised analyses, respectively. A two-dimensional gel-based approach analysis allowed us to identify that treatment with CM16 may downregulate four key proteins involved in glioma aggressiveness and associated with poor patient survival (HspB1, BTF3, PGAM1, and cofilin), while it may upregulate galectin-1 and Ebp1. Consistently with the protein synthesis inhibition properties of CM16, HspB1, Ebp1, and BTF3 exert known roles in protein synthesis. In conclusion, the downregulation of HspB1, BTF3, PGAM1 and cofilin bring new insights in CM16 antiproliferative effects, further supporting CM16 as an interesting protein synthesis inhibitor to combat glioma.

Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach.

Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer's disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.

In vivo biotinylated calpastatin improves the affinity purification of human m-calpain.

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies.

Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.

Secretion and functional display of fusion proteins through the curli biogenesis pathway.

Curli are functional amyloids expressed as fibres on the surface of Enterobacteriaceae. Contrary to the protein misfolding events associated with pathogenic amyloidosis, curli are the result of a dedicated biosynthetic pathway. A specialized transporter in the outer membrane, CsgG, operates in conjunction with the two accessory proteins CsgE and CsgF to secrete curlin subunits to the extracellular surface, where they nucleate into cross-beta strand fibres. Here we investigate the substrate tolerance of the CsgG transporter and the capability of heterologous sequences to be built into curli fibres. Non-native polypeptides ranging up to at least 260 residues were exported when fused to the curli subunit CsgA. Secretion efficiency depended on the folding properties of the passenger sequences, with substrates exceeding an approximately 2 nm transverse diameter blocking passage through the transport channel. Secretion of smaller passengers was compatible with prior DsbA-mediated disulphide bridge formation in the fusion partner, indicating that CsgG is capable of translocating non-linear polypeptide stretches. Using fusions we further demonstrate the exported or secreted heterologous passenger proteins can attain their native, active fold, establishing curli biogenesis pathway as a platform for the secretion and surface display of small heterologous proteins.

Alterations of the C-terminal end do not affect in vitro or in vivo activity of surfactant protein C analogs.

The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20 h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens.

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd(2+), Cu(2+), Zn(2+) and Pb(2+), with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.

Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems.

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn(2+) alone. The crystal structure of ZneB to 2.8 A resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the beta-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.

Ouabain-induced modifications of prostate cancer cell lipidome investigated with mass spectrometry and FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate modifications of prostate cancer PC-3 cell lipidome after exposure to sub-lethal concentrations of ouabain. FTIR spectroscopy offered an overview of the lipid classes present in the whole sample. The method is simple, label free and some features can be detected on entire cells. We compared the achievements of FTIR spectroscopy with data obtained by mass spectrometry (MS) on the same samples. It appears that FTIR spectroscopy could identify content variations in some lipid classes, e.g., these containing choline head groups such as phosphatidylcholine and sphingomyelin. MS analysis could confirm this result as indicated by principal component analysis and 2D heterocorrelation maps. FTIR spectra were also able to report changes in ester/choline/phosphate ratios characterizing lipid changes induced by ouabain. Furthermore, quantization of major lipid classes (PC, PE, PG, SM) could be obtained by curve fitting of the FTIR spectra. Yet, FTIR failed to resolve lipid classes for which the polar heads do not display specific IR features such as phosphatidylglycerol and cardiolipin.

Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.

Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas.

Melanomas display poor response rates to adjuvant therapies because of their intrinsic resistance to proapoptotic stimuli. This study indicates that such resistance can be overcome, at least partly, through the targeting of eEF1A elongation factor with narciclasine, an Amaryllidaceae isocarbostyril controlling plant growth. Narciclasine displays IC(50) growth inhibitory values between 30-100 nM in melanoma cell lines, irrespective of their levels of resistance to proapoptotic stimuli. Normal noncancerous cell lines are much less affected. At nontoxic doses, narciclasine also significantly improves (P=0.004) the survival of mice bearing metastatic apoptosis-resistant melanoma xenografts in their brain. The eEF1A targeting with narciclasine (50 nM) leads to 1) marked actin cytoskeleton disorganization, resulting in cytokinesis impairment, and 2) protein synthesis impairment (elongation and initiation steps), whereas apoptosis is induced at higher doses only (≥200 nM). In addition to molecular docking validation and identification of potential binding sites, we biochemically confirmed that narciclasine directly binds to human recombinant and yeast-purified eEF1A in a nanomolar range, but not to actin or elongation factor 2, and that 5 nM narciclasine is sufficient to impair eEF1A-related actin bundling activity. eEF1A is thus a potential target to combat melanomas regardless of their apoptosis-sensitivity, and this finding reconciles the pleiotropic cytostatic of narciclasine. -

Purification and crystallization of Phd, the antitoxin of the phd/doc operon.

The antitoxin Phd from the phd/doc module of bacteriophage P1 was crystallized in two distinct crystal forms. Crystals of His-tagged Phd contain a C-terminally truncated version of the protein and diffract to 2.20 A resolution. Crystals of untagged Phd purified from the Phd-Doc complex diffract to 2.25 A resolution. These crystals are partially merohedrally twinned and contain the full-length version of the protein.

Interplays between copper and Mycobacterium tuberculosis GroEL1.

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target.

Targeting an Intrinsically Disordered Protein by Covalent Modification.

Intrinsically disordered proteins (IDPs) play important roles in the regulation of cellular function and in disease, and thus they represent an important group of therapeutic targets. Yet, members of this "disorderome" have not yet been successfully targeted by drugs, primarily because traditional design principles cannot be applied to their highly dynamic, heterogeneous structural states. Binders developed against IDPs so far suffer from very weak binding and inability to act in a cellular context. Here, we describe a possible generic method for the targeting of IDPs via covalent modification, which could entail specific and strong binding and inhibitory potential, making such "warheads" reasonable starting points of drug-development efforts. We demonstrate this principle by addressing the cysteine-specific covalent modification of calpastatin, the IDP inhibitor of the calcium-dependent cysteine protease calpain. We describe the protocol for monitoring the covalent modification of the inhibitor, measuring the Ki of its inhibition and comparing it to the Kd of its interaction with the enzyme. Our premise is that the underlying principles can be easily adapted to screen for molecules targeting other, disease-related, IDPs in the future.

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility.

Phthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachidonyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with vancomycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates.

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin.

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.

Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75.

The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of L-glutamic acid and L-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations.

Affinity chromatography: a useful tool in proteomics studies.

Separation or fractionation of a biological sample in order to reduce its complexity is often a prerequisite to qualitative or quantitative proteomic approaches. Affinity chromatography is an efficient protein separation method based on the interaction between target proteins and specific immobilized ligands. The large range of available ligands allows to separate a complex biological extract in different protein classes or to isolate the low abundance species such as post-translationally modified proteins. This method plays an essential role in the isolation of protein complexes and in the identification of protein-protein interaction networks. Affinity chromatography is also required for quantification of protein expression by using isotope-coded affinity tags.

Data in support of a harmine-derived beta-carboline in vitro effects in cancer cells through protein synthesis.

A harmine-derived beta-carboline, CM16, inhibits cancer cells growth through its effects on protein synthesis, as described in "A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis" (Carvalho et al., 2017)[1]. This data article provides accompanying data on CM16 cytostatic evaluation in cancer cells as well as data related to its effects on transcription and translation. After confirming the cytostatic effect of CM16, we investigated its ability to arrest the cell cycle in the glioma Hs683 and SKMEL-28 melanoma cell lines but no modification was evidenced. According to the global protein synthesis inhibition induced by CM16 [1], transcription phase, a step prior to mRNA translation, evaluated by labelled nucleotide incorporation assay was not shown to be affected under CM16 treatment in the two cell lines. By contrast, mRNA translation and particularly the initiation step were shown to be targeted by CM16 in [1]. To further decipher those effects, we established herein a list of main actors in the protein synthesis process according to literature survey for comparative analysis of cell lines displaying different sensitivity levels to CM16. Finally, one of these proteins, PERK, a kinase regulating eIF2-α phosphorylation and thereby activity, was evaluated under treatment with CM16 in a cell-free system.