High-resolution structure of a papaya plant-defense barwin-like protein solved by in-house sulfur-SAD phasing.

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Šwavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central ß-barrel of six strands surrounded by several α-helices and loops. The ß-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases.

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex.

A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 A, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-D-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2(1), with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 A, beta = 95.33 degrees and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 A. Structure refinement is currently in progress.

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.

Chemical constituents, cytotoxic, antifungal and antimicrobial properties of Centaurea diluta Ait. subsp. algeriensis (Coss. & Dur.) Maire.

OBJECTIVE: To investigate the chemical composition of a moderately polar extract (CHCl3 soluble part of the MeOH-H2O extract) obtained from the aerial parts (leaves and flowers) of Centaurea diluta Ait. subsp. algeriensis (Coss. & Dur.) Maire, a species endemic to Algeria and Morocco on which no reports are available to date. To evaluate in vitro the cytotoxic, antifungal and antimicrobial activities of this extract and the cytotoxic and antimicrobial activities of its isolated secondary metabolites. METHODS: The cytotoxic effects of the extract were investigated on 3 human cancer cell lines i.e. the A549 non-small-cell lung carcinoma (NSCLC), the MCF7 breast adenocarcinoma and the U373 glioblastoma using a MTT colorimetric assay. Biological data allowed to guide the fractionation of the extract by separation and purification on silica gel 60 (CC and TLC). The isolated compounds which were characterized by spectral analysis, mainly HR-ESIMS, HR-EIMS, UV and NMR experiments ((1)H, (13)C, COSY, ROESY, HSQC and HMBC) and comparison of their spectroscopic data with those reported in the literature, were evaluated for cytotoxic activities on six cancer cell lines (A549, MCF7, U373, Hs683 human glioma, PC3 human prostate and B16-F10 murine melanoma). The direct and indirect antibacterial and antifungal activities were determined using microdilution methods for the raw extract and TLC-bioautography and microdilution methods against standard and clinical strains for the isolated compounds. RESULTS: The raw extract reduced cell viability with IC50s of 27, 25 and 21 µg/mL on A549, MCF7 and U373, respectively. Five secondary metabolites: two phenolic compounds (vanillin 1, paridol 3), a lignan [(-)-arctigenin 2] and two flavonoid aglycones (eupatilin 4 and jaceosidin 5), were then isolated from this extract. Moderate cytotoxic effects were observed for (-)-arctigenin 2 (IC50s: 28 and 33 µM on Hs683 and B16-F10, respectively), eupatilin 4 (IC50s: 33 and 47 µM on B16-F10 and PC3, respectively) and jaceosidin 5 (IC50s: 32 and 40 µM on PC3 and B16-F10, respectively). CONCLUSIONS: All the isolated compounds were described for the first time from this species. Although inactive against 7 tested microorganisms (fungi, bacteria and yeast, human or plant pathogens), the raw extract was able to potentiate the effect of beta-lactam antibiotics on methicillin-resistant Staphylococcus aureus (MRSA), reducing the minimal inhibitory concentrations (MICs) by a factor of 2-32-fold. No synergy was found between the extract and streptomycin. From the five isolated compounds only jaseosidin 5 showed a moderate antimicrobial activity.

Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume.

Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms.

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination.

A chip format sandwich-type immunoassay based on Nanobodies(®) (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20 min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200 µg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized.

Hypothesis/review: the structural basis of sweetness perception of sweet-tasting plant proteins can be deduced from sequence analysis.

Human perception of sweetness, behind the felt pleasure, is thought to play a role as an indicator of energy density of foods. For humans, only a small number of plant proteins taste sweet. As non-caloric sweeteners, these plant proteins have attracted attention as candidates for the control of obesity, oral health and diabetic management. Significant advances have been made in the characterization of the sweet-tasting plant proteins, as well as their binding interactions with the appropriate receptors. The elucidation of sweet-taste receptor gene sequences represents an important step towards the understanding of sweet taste perception. However, many questions on the molecular basis of sweet-taste elicitation by plant proteins remain unanswered. In particular, why homologues of these proteins do not elicit similar responses? This question is discussed in this report, on the basis of available sequences and structures of sweet-tasting proteins, as well as of sweetness-sensing receptors. A simple procedure based on sequence comparisons between sweet-tasting protein and its homologous counterparts was proposed to identify critical residues for sweetness elicitation. The open question on the physiological function of sweet-tasting plant proteins is also considered. In particular, this review leads us to suggest that sweet-tasting proteins may interact with taste receptor in a serendipity manner.

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

BACKGROUND: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. RESULTS: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a ß-hairpin pointed towards the substrate binding cleft. The invariant ß-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. CONCLUSIONS: The seven families and subfamilies of the lysozyme superfamily all have in common a ß-hairpin structure which displays a family-specific sequence motif. These GH ß-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.

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.

Structural characterization of the papaya cysteine proteinases at low pH.

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.