Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases-3

E body homogenates of eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. : Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).<p><b>Copyright information:</b></p><p>Taken from "Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases"</p><p>http://www.parasitesandvectors.com/content/1/1/7</p><p>Parasites & Vectors 2008;1():7-7.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2289814.</p><p></p

Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases-1

Lective peptide substrates (structure of the fluorogenic substrates is indicated). The activity for individual substrates was suppressed in the presence of selective peptidase inhibitors to obtain diagnostic responses indicative of a protease type. Values are expressed as percent inhibition of the control activities. The identified major activities (Target) correspond to papain-type peptidases cathepsin B, L and C (CathB, CathL and CathC, respectively), cathepsin D-like aspartic peptidase (CathD) and asparaginyl endopeptidase (AE). The assay was performed at pH 4.0, an optimum pH for hemoglobin degradation by the gut extract. The activity of AE and CathL was measured in the presence of CA-074 inhibitor to prevent an interference with the activity of CathB. The error bars indicate standard deviations of the mean of triplicates.<p><b>Copyright information:</b></p><p>Taken from "Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases"</p><p>http://www.parasitesandvectors.com/content/1/1/7</p><p>Parasites & Vectors 2008;1():7-7.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2289814.</p><p></p

Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases-0

Orescently labeled AMC-hemoglobin was digested with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.<p><b>Copyright information:</b></p><p>Taken from "Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases"</p><p>http://www.parasitesandvectors.com/content/1/1/7</p><p>Parasites & Vectors 2008;1():7-7.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2289814.</p><p></p

Quantum Mechanics-Based Scoring Rationalizes the Irreversible Inactivation of Parasitic <i>Schistosoma mansoni</i> Cysteine Peptidase by Vinyl Sulfone Inhibitors

The quantum mechanics (QM)-based scoring function that we previously developed for the description of noncovalent binding in protein–ligand complexes has been modified and extended to treat covalent binding of inhibitory ligands. The enhancements are (i) the description of the covalent bond breakage and formation using hybrid QM/semiempirical QM (QM/SQM) restrained optimizations and (ii) the addition of the new Δ<i>G</i>_cov′ term to the noncovalent score, describing the “free” energy difference between the covalent and noncovalent complexes. This enhanced QM-based scoring function is applied to a series of 20 vinyl sulfone-based inhibitory compounds inactivating the cysteine peptidase cathepsin B1 of the Schistosoma mansoni parasite (SmCB1). The available X-ray structure of the SmCB1 in complex with a potent vinyl sulfone inhibitor K11017 is used as a template to build the other covalently bound complexes and to model the derived noncovalent complexes. We present the correlation of the covalent score and its constituents with the experimental binding data. Four outliers are identified. They contain bulky R₁′ substituents structurally divergent from the template, which might induce larger protein rearrangements than could be accurately modeled. In summary, we propose a new computational approach and an optimal protocol for the rapid evaluation and prospective design of covalent inhibitors with a conserved binding mode

Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases-2

Zymes. The horizontal bar represents a distance of 0.1 substitutions per site. Numbers at the branches display bootstrap support. : (AAH95408), (P00787), (ABO26563), (BAF43801), (P25792), (P43157); : (M20496), (AAH63175), (ABO26562), (AF227957), (CathL1 (Z32529), CathL2 (U07345)), (CathL1 (U38476), CathL2 (U38475)), (P25779); : (X87212), (D90404), (ABV29335), (Z32531), (U77932).<p><b>Copyright information:</b></p><p>Taken from "Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases"</p><p>http://www.parasitesandvectors.com/content/1/1/7</p><p>Parasites & Vectors 2008;1():7-7.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2289814.</p><p></p

Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases-4

Orescently labeled AMC-hemoglobin was digested with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.<p><b>Copyright information:</b></p><p>Taken from "Profiling of proteolytic enzymes in the gut of the tick reveals an evolutionarily conserved network of aspartic and cysteine peptidases"</p><p>http://www.parasitesandvectors.com/content/1/1/7</p><p>Parasites & Vectors 2008;1():7-7.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2289814.</p><p></p

Detailed micrograph of SmPOP localization in the tegument of adult <i>S</i>. <i>mansoni</i>.

<p>The tissue section was probed with anti-SmPOP IgG followed by an anti-rabbit IgG Alexa 594-labeled secondary antibody (red). DAPI was used to label the nuclear DNA (blue). The left image shows merged fluorescent channels; on the right, the fluorescent signal is merged with differential interference contrast imaging. A red fluorescent signal is found in the parenchyma (Pa) and tegument (Teg), but is absent from the gastrodermis (Gd), gut lumen (the asterisks) and muscular tissue (Mu). Male worms (M) show a stronger immuno-reactivity than female worms (F). Note the difference in the signal intensity on the tegument of the male compared to the female (the connected arrowheads). Scale bar = 20 μm.</p

Activity and transcriptional profiling of SmPOP in the developmental stages of <i>S</i>. <i>mansoni</i>.

<p>(A) The expression of SmPOP was evaluated by quantitative RT-PCR. mRNA transcriptional levels are presented as the percentage of expression relative to the constitutively expressed <i>S</i>. <i>mansoni</i> cytochrome oxidase I (SmCOX I). The mean values ± S.D. of three replicates are given. (B) SmPOP activities were measured in protein extracts of the developmental stages (except sporocysts not available in sufficient amount and purity) using a kinetic assay with the fluorogenic substrate Z-Gly-Pro-AMC at pH 8.0. POP activities (sensitive to inhibition by the specific POP inhibitor Z-Pro-Pro-CHO) are expressed in relative fluorescence units (RFU/s) and normalized to protein content.</p

SmPOP is localized to the tegument and parenchyma of adult <i>S</i>. <i>mansoni</i>.

<p>Semi-thin sections of adult male and female <i>S</i>. <i>mansoni</i> were probed with an anti-SmPOP IgG (A–F) or a pre-immune IgG (G, H) followed by reaction with an anti-rabbit IgG Alexa 594-labeled secondary antibody (red). DAPI was used to label the nuclear DNA (blue); female vitellaria are characterized by strong autofluorescence in the green spectrum (H). The left column shows merged fluorescent channels; in the right column, the signal is merged with differential interference contrast (except in H). Male worms (M) incubated with anti-SmPOP show a stronger immune-reactivity than female worms (F) (micrographs A and B). A red fluorescent signal is found in the parenchyma and tegument, but it is absent from the gut (the asterisks in A, B, E and F) and muscular tissue (Mu, micrographs C-F). In male worms the signal is found accumulated in the tubercles of the dorsal tegument (the arrows in C and D) and also outlines the gynaecophoral canal. Note the difference in signal intensity between the male and female tegument (the connected arrowheads in E and F). Only faint background fluorescence could be detected in the red spectrum in the negative control probed with pre-immune IgG (the micrographs G and H). The scale bar in C and D represents 20 μm; in A, B, E–H, 50 μm.</p

A three-dimensional homology model of SmPOP.

<p>(A) A ribbon representation of the overall SmPOP structure showing the β-propeller domain (cyan) and the catalytic domain (pink); the N-terminal segment is colored green. The active site containing the catalytic triad Ser556, Asp643 and His682 (yellow) is located at the interface of the two domains. (B) A superposition of the SmPOP model (green) and the porcine POP crystal structure (pink with the PDB code 1QFS) in a cylinder representation. (C) A view from the top of the SmPOP model (the β-propeller domain (cyan, ribbon representation) controls access to the active site of the catalytic domain (the pink surface) indicated by the catalytic triad residues (the yellow surface and sticks); the N-terminal segment is shown as the green surface. (D) A surface representation of the SmPOP active site located in the catalytic domain (the pink surface). The covalently-bound inhibitor Z-Pro-Pro-CHO is depicted in the ball-and-stick representation (carbon atoms in blue, oxygen in red and nitrogen in light blue). The catalytic-domain residues forming contacts with the inhibitor are highlighted as the magenta surface; the catalytic triad residues are represented by the yellow surface/sticks. The β-propeller domain is shown as a cyan ribbon, the residues interacting with the inhibitor as cyan sticks.</p