Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

Oxidative Transformation of Dihydroflavonols and Flavan-3-ols by Anthocyanidin Synthase from Vitis vinifera.

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate-cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C3-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.

Design of Polysaccharide-b-Elastin-Like Polypeptide Bioconjugates and Their Thermoresponsive Self-Assembly.

The advantageous biological properties of polysaccharides and precise stimuli-responsiveness of elastin-like polypeptides (ELPs) are of great interest for the design of polysaccharide- and polypeptide-based amphiphilic block copolymers for biomedical applications. Herein, we report the synthesis and characterization of a series of polysaccharide-block-ELP copolymers, containing two biocompatible and biodegradable blocks coupled via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The resulting bioconjugates are capable of self-assembling into well-defined nanoparticles in aqueous solution upon raising the solution temperature above a specific transition temperature (Tt)-a characteristic of the ELP moiety. To the best of our knowledge, this is the first study where polysaccharides were combined with a stimuli-responsive ELP for the preparation of thermosensitive self-assemblies, providing insight into novel pathways for designing bioinspired stimuli-responsive self-assemblies for biomedical applications.

Assessing Interactions between Helical Aromatic Oligoamide Foldamers and Protein Surfaces: A Tethering Approach.

Helically folded aromatic foldamers may constitute suitable candidates for the ab initio design of ligands for protein surfaces. As preliminary steps toward the exploration of this hypothesis, a tethering approach was developed to detect interactions between a protein and a foldamer by confining the former at the surface of the latter. Cysteine mutants of two therapeutically relevant enzymes, CypA and IL4, were produced. Two series of ten foldamers were synthesized bearing different proteinogenic side chains and either a long or a short linker functionalized with an activated disulfide. Disulfide exchange between the mutated cysteines and the activated disulfides yielded 20 foldamer-IL4 and 20 foldamer-CypA adducts. Effectiveness of the reaction was demonstrated by LC-MS, by MS analysis after proteolytic digestion, and by 2D NMR. Circular dichroism then revealed diastereoselective interactions between the proteins and the foldamers confined at their surface which resulted in a preferred handedness of the foldamer helix. Helix sense bias occurred sometimes with both the short and the long linkers and sometimes with only one of them. In a few cases, helix handedness preference is found to be close to quantitative. These cases constitute valid candidates for structural elucidation of the interactions involved.

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.

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.

Recombinant production and purification of short hydrophobic Elastin-like polypeptides with low transition temperatures.

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELPs (VPGIG)n with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 °C, 12.4 °C and 11.7 °C, respectively.

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.

Species identification of ivory and bone museum objects using minimally invasive proteomics.

Ivory is a highly prized material in many cultures since it can be carved into intricate designs and have a highly polished surface. Due to its popularity, the animals from which ivory can be sourced are under threat of extinction. Identification of ivory species is not only important for CITES compliance, it can also provide information about the context in which a work was created. Here, we have developed a minimally invasive workflow to remove minimal amounts of material from precious objects and, using high-resolution mass spectrometry-based proteomics, identified the taxonomy of ivory and bone objects from The Metropolitan Museum of Art collection dating from as early as 4000 B.C. We built a proteomic database of underrepresented species based on exemplars from the American Museum of Natural History, and proposed alternative data analysis workflows for samples containing inconsistently preserved organic material. This application demonstrates extensive ivory species identification using proteomics to unlock sequence uncertainties, e.g., Leu/Ile discrimination.

Solution behavior and encapsulation properties of fatty acid-elastin-like polypeptide conjugates.

Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery. Elastin-like polypeptides (ELPs) are good candidates for these applications because they are biosourced, biocompatible and biodegradable. With the aim of developing ELP-based micelles for drug delivery applications we have synthesized 15 acyl-ELP compounds by conjugating myristic, palmitic, stearic, oleic or linoleic acid to the N-terminus of three ELPs differing in molar mass. The ELP-fatty acid conjugates have interesting solution behavior. They form micelles at low temperatures and aggregate above the cloud point temperature (Tcp). The critical micelle concentration depends on the fatty acid nature while the micelle size is mainly determined by the ELP block length. We were able to show that ELPs were better hydrated in the micelles than in their individual state in solution. The micelles are stable in phosphate-buffered saline at temperatures below the Tcp, which can vary between 20 °C and 38 °C depending on the length or hydrophilicity of the ELP. Acyl-ELP micelles were loaded with the small hydrophobic molecule Nile red. The encapsulation efficiency and release kinetics showed that the best loading conditions were achieved with the largest ELP conjugated to stearic acid.

Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in inflammatory bowel diseases.

Human secretory immunoglobulins (SIg) A1 and SIgA2 guide mucosal responses toward tolerance or inflammation, notably through reverse-transcytosis, the apical-to-basal transport of IgA2 immune complexes via M cells of gut Peyer's patches. As such, the maintenance of a diverse gut microbiota requires broad affinity IgA and glycan-glycan interaction. Here, we asked whether IgA1 and IgA2-microbiota interactions might be involved in dysbiosis induction during inflammatory bowel diseases. Using stool HPLC-purified IgA, we show that reverse-transcytosis is abrogated in ulcerative colitis (UC) while it is extended to IgA1 in Crohn's disease (CD). 16S RNA sequencing of IgA-bound microbiota in CD and UC showed distinct IgA1- and IgA2-associated microbiota; the IgA1+ fraction of CD microbiota was notably enriched in beneficial commensals. These features were associated with increased IgA anti-glycan reactivity in CD and an opposite loss of reactivity in UC. Our results highlight previously unknown pathogenic properties of IgA in IBD that could support dysbiosis.

The epimerase activity of anthocyanidin reductase from Vitis vinifera and its regiospecific hydride transfers.

Anthocyanidin reductase (ANR) from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins producing a mixture of (2S,3R)- and (2S,3S)-flavan-3-ols. At pH 7.5 and 30 degrees C, the first hydride transfer to anthocyanidin is irreversible, and no intermediate is released during catalysis. ANR reverse activity was assessed in the presence of excess NADP(+). Analysis of products by reverse phase and chiral phase HPLC demonstrates that ANR acts as a flavan-3-ol C(3)-epimerase under such conditions, but this is only observed with 2R-flavan-3-ols, not with 2S-flavan-3-ols produced by the enzyme in the forward reaction. In the presence of deuterated coenzyme 4S-NADPD, ANR transforms anthocyanidins into dideuterated flavan-3-ols. The regiospecificity of deuterium incorporation into catechin and afzelechin - derived from cyanidin and pelargonidin, respectively - was analyzed by liquid chromatography coupled with electro- spray ionization-tandem mass spectrometry (LC/ESI-MS/MS), and it was found that deuterium was always incorporated at C(2) and C(4). We conclude that C(3)-epimerization should be achieved by tautomerization between the two hydride transfers and that this produces a quinone methide intermediate which serves as C(4) target of the second hydride transfer, thereby avoiding any stereospecific modification of carbon 3. The inversion of C(2) stereochemistry required for 'reverse epimerization' suggests that the 2S configuration induces an irreversible product dissociation.

Analysis of lipid-oligonucleotide conjugates by cyclodextrin-modified capillary zone electrophoresis.

Lipid-oligonucleotide (LONs) based bioconjugates represent an emerging class of therapeutic agents, allowing the delivery of therapeutic oligonucleotide sequences. The LON development requests accurate and efficient analytical methods. In this contribution, LON analysis methods were developed in cyclodextrin-modified capillary zone electrophoresis (CD-CZE). The LONs selected in this study feature different structures, including i) the oligonucleotide length (from 10 to 20 nucleotides), ii) the inter-nucleotide linkage chemistry (phosphodiester PDE or phosphorothioate PTO), and iii) the lipidic part: single- (LONsc) or double-chain (LONdc) lipids. In CD-CZE, the effect of several parameters on the electrophoretic peaks was investigated (buffer, CD, and capillary temperature). The binding interaction between LON and Me-ß-CD was studied in affinity capillary electrophoresis and revealed a 1:1 LON:CD complex. Non-linear regression and three usual linearization methods (y-reciprocal, x-reciprocal, and double-reciprocal) were used to determine the binding constants (K values of 2.5.104 M-1 and 2.0.104 M-1 for LON PDE and LON PTO, respectively). Quantitative methods with good performances and analysis time lower than 5 min were achieved. Importantly, the developed analysis allows a separation between the i) full-length sequence LONs and their truncated sequences, (n-1), (n-2), and (n-4)-mers and ii) LONsc, LONdc and their corresponding unconjugated oligonucleotides. This work highlights the interest of CD-CZE methods for LON analysis.

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.

Asymmetric hydroxylative phenol dearomatization through in situ generation of iodanes from chiral iodoarenes and m-CPBA.

'I' is all the hype: A twofold excess of iodoarene in the title reaction leads to ortho-quinols in good yields, whereas organocatalytic versions of this reaction enable subsequent epoxidation in a regio- and diastereoselective fashion. Chiral iodobiarenes led to enantioselectivities up to 50 % ee. m-CPBA = meta-chloroperoxybenzoic acid.

Oxidative Transformation of Leucocyanidin by Anthocyanidin Synthase from Vitis vinifera Leads Only to Quercetin.

Anthocyanidin synthase from Vitis vinifera ( VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2 R,3 S,4 S- cis-leucocyanidin, into 2 R,4 S-flavan-3,3,4-triol ([M + H]+, m/ z 323) and quercetin. The C3-hydroxylation product 2 R,4 S-flavan-3,3,4-triol is first produced and its C3,C4-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C3-hydroxylation leading to a 4-keto-2 R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2 R,3 S,4 R- trans-leucocyanidin, is similarly transformed into quercetin upon C3,C4-dehydration, but unlike 3,4- cis-leucocyanidin, it also undergoes some C2,C3-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C4 to give traces of cyanidin. Overall, the C3,C4- trans isomer of leucocyanidin is transformed into 2 R,4 R-flavan-3,3,4-triol (M + 1, m/ z 323), (+)-DHQ, (-)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4- cis-leucocyanidin- and 3,4- trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C3-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.

The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment.

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2TM22, or a peptide mutated at the central residues G100/C103 (VAMP2TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2TM22, in terms of α-helices and ß-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G100/C103 within the transmembrane domain during lipid rearrangements in membrane fusion.

Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications.

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis.

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 A resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.

Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct MS/MS Fragmentation Patterns.

(+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin synthesized by reduction of (+)-dihydroquercetin with NaBH4, and structures of the two stereoisomers purified by C18- and phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in aqueous solution at -20 °C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways, which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode, which involves a loss of hydroxyl group at either C3 or C4 for the 3,4-cis isomer but only at C3 for the 3,4-trans isomer. Tandem mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers.