Guy Vandenbussche

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 IC50 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.—Van Goietsenoven, G., Hutton, J., Becker, J.‐P., Lallemand, B., Robert, F., Lefranc, F., Pirker, C., Vandenbussche, G., Van Antwerpen, P., Evidente, A., Berger, W., Prevost, M., Pelletier, J., Kiss, R., Goss Kinzy, T., Kornienko, A., Mathieu, V. Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas. FASEB J. 24, 4575–4584 (2010). www.fasebj.org

Role of the carboxy terminus of Escherichia coli FtsA in self-interaction and cell division.

The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the ftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsADelta1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsADelta27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsADelta27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

The 21-residue surfactant peptide (LysLeu4)4Lys(KL4) is a transmembrane α-helix with a mixed nonpolar/polar surface

The 21‐residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4) has been synthesized and analyzed regarding its secondary structure and orientation in lipid environments. Fourier transform infrared and circular dichroism spectroscopy shows that the peptide exhibits approximately 80% α‐helical content both in dodecylphosphocholine micelles and in 1,2‐dipalmitoyl‐glycero‐3‐phosphocholine (DPPC)/phosphatidylglycerol (PG) 7:3 (w/w) bilayers. The positively charged lysine residues are evenly distributed over the entire, otherwise nonpolar, circumference of the helix. This is in sharp contrast to the uneven distribution of polar and nonpolar residues in amphipathic helices. Fourier transform infrared spectroscopy of the peptide inserted in DPPC/PG bilayers shows that the helical axis is oriented parallel to the lipid acyl chains. These data do not support a previous hypothesis that the KL4 peptide interacts with peripheral parts of a phospholipid monolayer and mimics the pulmonary surfactant protein SP‐B, which is composed of several amphipathic α‐helices. KL4 accelerates the spreading of phospholipid mixtures at an air/water interface but does so less efficiently than other transmembranous helical polypeptides studied.

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 Zn2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-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.

Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Conformational changes in a bacterial multidrug transporter are phosphatidylethanolamine-dependent.

Abstract.LmrP is an electrogenic H+/drug antiporter that extrudes a broad spectrum of antibiotics. Five carboxylic residues are implicated in drug binding (Asp142 and Glu327) and proton motive force-mediated restructuring (Asp68, Asp128 and Asp235). ATR-FTIR (Attenuated Total Reflection — Fourier Transform Infrared) and tryptophan quenching experiments revealed that phosphatidylethanolamine (PE) is required to generate the structural intermediates induced by ionization of carboxylic residues. Surprisingly, no ionization-induced conformational changes were detectable in the absence of PE, suggesting either that carboxylic acid residues do not ionize or that ionization does not lead to any conformational change. The mean pKa of carboxylic residues evaluated by ATR-FTIR spectroscopy was 6.5 for LmrP reconstituted in PE liposomes, whereas the pKa calculated in the absence of PE was 4.6. Considering that 16 of the 19 carboxylic residues are located in the extramembrane loops, the pKa values obtained in the absence and in the presence of PE suggest that the interaction of the loop acid residues with the membrane interface depends on the lipid composition.

Very low surfactant protein C contents in newborn Belgian White and Blue calves with respiratory distress syndrome

We have studied a respiratory distress syndrome (RDS) occurring in newborn calves of the Belgian White and Blue (BWB) breed that represents the large majority of beef cattle in Belgium. Pulmonary surfactant isolated from 14 BWB newborn calves that died from RDS and from 7 healthy controls was analysed for composition and surface activity. An extremely low content or, in some instances, an absence of surfactant protein C (SP-C) was detected in the RDS samples by Western blotting and differential amino acid analysis [0.03+/-0.01% (w/w) relative to total phospholipids, compared with 0.39+/-0.06% for healthy controls (means+/-S.E.M., P < 0.001)]. The contents of surfactant protein B (SP-B) were similar in RDS and control samples. The crude surfactant samples isolated from RDS calves had higher ratios of total protein to total phospholipid, altered phospholipid profiles and lower SP-A contents. Both crude and organic extracts of RDS surfactant samples showed increased dynamic surface tension compared with healthy controls when evaluated with a pulsating-bubble surfactometer. The addition of purified SP-C to organic extracts of RDS surfactant samples lowered surface tension. Strongly decreased levels of mature SP-C associated with fatal RDS and altered surface activity in vitro have, to the best of our knowledge, not been previously reported. The mechanisms underlying RDS and the decrease in SP-C in BWB calves remain to be established.

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.

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a DeltaG(water)(0) of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.

Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence

Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.