Katell Bathany

Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina

The [Het‐s] infectious element of the fungus Podospora anserina is a prion protein involved in a genetically controlled cell death reaction termed heterokaryon incompatibility. Previous analyses indicate that [Het‐s] propagates as a self‐perpetuating amyloid aggregate. The HET‐s protein is 289 amino acids in length. Herein, we identify the region of the HET‐s protein that is responsible for amyloid formation and prion propagation. The region of HET‐s spanning residues 218–289 forms amyloid fibers in vitro and allows prion propagation in vivo. Conversely, a C‐terminal deletion in HET‐s prevents amyloid aggregation in vitro and prion propagation in vivo, and abolishes the incompatibility function. In the soluble form of HET‐s, the region from residue 1 to 227 forms a well‐folded domain while the C‐terminal region is highly flexible. Together, our data establish a domain structure–function relationship for HET‐s amyloid formation, prion propagation and incompatibility activity.

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Δ5-sterols are replaced by 9β,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Δ5-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.

Structure and epimerase activity of anthocyanidin reductase from Vitis vinifera.

Together with leucoanthocyanidin reductase, anthocyanidin reductase (ANR) is one of the two enzymes of the flavonoid-biosynthesis pathway that produces the flavan-3-ol monomers required for the formation of proanthocyanidins or condensed tannins. It has been shown to catalyse the double reduction of anthocyanidins to form 2R,3R-flavan-3-ols, which can be further transformed to the 2S,3R isomers by non-enzymatic epimerization. ANR from grape (Vitis vinifera) was expressed in Escherichia coli and purified. Unexpectedly, RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme produces a 50:50 mixture of 2,3-cis and 2,3-trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S- and 2S,3R-flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (-)-catechin, when cyanidin is used as the substrate of the reaction. The first three-dimensional structure of ANR is described at a resolution of 2.2 A and explains the inactivity of the enzyme in the presence of high salt concentrations.

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air-water interface

Cateslytin (bCGA 344RSMRLSFRARGYGFR358), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin.

Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry

In the context of proteome analysis, matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl‐tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI‐MS for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post‐source decay analysis for confident protein identification.

Lipid oligonucleotide conjugates as responsive nanomaterials for drug delivery

We report Lipid OligoNucleotide conjugates (LONs) bearing either two or three hydrophobic chains. LONs self-assemble into micellar aggregates, which provide a suitable reservoir for hydrophobic drugs such as paclitaxel. Our results demonstrate that the composition of the LONs both in terms of the lipid and the oligonucleotide sequence impacts their ability to host lipophilic molecules. Interestingly, binding of the complementary oligonucleotide selectively induces the release of part of the drug payload of the aggregates. These LON based micelles, which efficiently host hydrophobic drugs, represent an original stimuli-responsive drug delivery system.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass.

Selective Tuning of Elastin-like Polypeptide Properties via Methionine/Oxidation

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.

2′-O-Appended Polyamines that Increase Triple-Helix-Forming Oligonucleotide Affinity are Selected by Dynamic Combinatorial Chemistry

The sequence-specific recognition of double-stranded DNA by triplex-forming oligonucleotides (TFOs) has potential application as therapeutics in the antigene strategy as well as tools in molecular biology. TFOs bind to the major groove of DNA either in a parallel or antiparallel orientation according to their base composition. TFOs composed of pyrimidine bases bind parallel to the purine strand of oligopurine–oligopyrimidine duplexes through T-AT and C-GC triplet formation. However, the use of TFOs under physiological pH and ionic strength is hampered by their weak binding to DNA duplexes mainly due to the requirement for cytosine protonation and charge repulsion between the three negatively charged strands. Spermine and related polyamines are largely protonated at physiological pH and are known to promote triplex stabilization both upon external addition as well as upon conjugation at the 5’-terminus, C5 of dU, and C4 of 5-methyl-dC. In contrast, attachment of spermine on a 2’-position in a TFO was found to have a deleterious effect on triplex stability. In this case, replacement of one internal dT unit by an ara U 2’-phosphorylpropylspermine within an oligo dT was found to abolish its capacity to form a triplex with an oligo-dA·oligo-dT duplex; this suggests that tethering position, ara C2’, or/and the linker phosphorylpropyl was inappropriate. However, Cuenoud et al. showed later that TFOs containing 2’-O-aminoethyl ribonucleotides (2’-AE-TFOs) formed stabilized triplexes owing to dual recognition of DNA targets by base–base contacts and concomitant salt-bridge formation between positively charged ammonium groups on the TFO and DNA phosphates. This dual recognition approach would be further improved if amino groups present on 2’ positions of a 2’-AE-TFO were substituted by polyamines, thus generating 2’-polycationic chains that are able to strongly interact with several DNA phosphate groups and bring additional stabilization to the triplex. In this context, dynamic combinatorial chemistry (DCC) appears to be a method of choice for the identification of such 2’-O-appended polyamines. DCC has attracted increasing interest over recent years as an alternative approach to traditional combinatorial chemistry that combines in a single step the library build-up and screening processes. DCC involves the use of reversible reactions between different building blocks to generate an equilibrating mixture of compounds that is able to respond through noncovalent interactions to the addition of a target molecule. The preferential binding of one member of the mixture to the target induces a shift in the equilibrium towards that particular compound. Thus DCC offers in situ screening of the combinatorial library simply by comparing its composition in the absence and presence of the target. DCC experiments have been performed by using various biological targets including nucleic acids. In previous studies, we have established that DCC can be used to identify covalently appended small molecules that stabilize oligonucleotide complexes. f] For that purpose, equilibrating imines formed from 2’-aminonucleotide incorporated into an oligonucleotide ligand and a small set of aldehydes were submitted to the template effect of a nucleic acid target. These studies were carried out in the context of DNA and RNA duplexes as well as an RNA–RNA kissing complex. In each case, after reduction of the imines, a chemically stable conjugated ligand with an increased affinity for its target was identified that corresponded to the most amplified compound. Here, by using an “inverted” imine reaction, we report an application of DCC for the screening of various amines and polyamines for their ability upon reaction with a 2’-linked aldehyde group present in an internal ribonucleoside unit of a TFO to stabilize the triplex formed with a DNA target. Eleven-base-long 2’-O-methyl TFO 1 bearing a central 2’-O(2-oxoethyl)uridine and able to form a parallel triplex with the stem of DNA hairpin 2 was synthesized (Scheme 1 A). TFO 1 was obtained by periodate-mediated oxidation of precursor 2’O-Me oligonucleotide containing a 2’-O-(2,3-dihydroxypropyl)uridine in position 6 and synthesized by the phosphoramidite method. The fully protected phosphoramidite of this latter nucleoside was synthesized (Scheme 2) by using a slightly modified procedure as described by Zatsepin et al. Starting from uridine, the less-expensive 3’,5’-di-tert-butyl disiloxane protecting group was preferred to Markewicz’s reagent; protection of the 3-N position by pivaloyloxymethyl (Pom) group was necessary for further selective 2’-O allylation. Palladiumassisted allylation proceeded in good yield, and the vic-diol was subsequently produced through oxidation by osmium tetroxide/4-methylmorpholine N-oxide (NMO). The diol was protected through acetylation, and the resulting uridine derivative was 3’-O phosphitylated. [a] Dr. L. Az ma , Dr. B. Rayner U869, INSERM 146 rue L o Saignat, 33076 Bordeaux (France) Fax: (+ 33) 5-57-57-45-65 E-mail : bernard.rayner@inserm.fr [b] Dr. L. Az ma , Dr. K. Bathany , Dr. B. Rayner Department of Technology for Health, University of Bordeaux 146 rue L o Saignat, 33076 Bordeaux (France) [c] Dr. K. Bathany European Institute of Chemistry and Biology 2 rue Robert Escarpit, 33607 Pessac (France) [d] Dr. K. Bathany Present address: Centre de G nomique Fonctionnelle 146 rue L o Saignat, 33076 Bordeaux (France) Fax: (+ 33) 5-57-57-16-84 Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000538.

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.