32 research outputs found

    TMPRSS6 isoforms 3 and 4 reduce isoform 2 activity.

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    A) TMPRSS6 isoforms representation adapted from our previous publication [1] licensed under CC BY 3.0. B) Expression of TMPRSS6 V5-tagged isoforms 2, 3 and 4 in transfected HEK293 cells assessed by western blotting against V5. C) Proteolytic activity was measured in the cell medium of HEK293 cells transfected with one or two TMPRSS6 V5-tagged isoforms. Boc-QAR-AMC (200 μM) fluorogenic substrate cleavage was monitored. Results are presented as relative activity over TMPRSS6 isoform 2 (TMPRSS6-2) activity. Statistical significance was assessed using one sample T test, *p < 0.0002 (n = 5).</p

    TMPRSS6 isoforms engage in homo- and hetero-interactions.

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    HEK293 cells were co-transfected with V5- and HA-tagged TMPRSS6 isoforms. Homo- and hetero-interaction of TMPRSS6 isoforms were assessed by immunoprecipitation (IP) of cell lysate (CL) using an anti-V5 antibody. Isoform immunoprecipitation was detected using anti-V5 and anti-HA antibodies. Equal amount of CL was loaded on SDS-polyacrylamide gels and cell GAPDH was used as a loading control (n = 3).</p

    TfR1 cleavage controls.

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    TfR1-HA was transfected either with V5-tagged TMPRSS6 isoform 2 WT (active, TMPRSS6-2-WT-V5), catalytically inactivated TMPRSS6 isoform 2 (TMPRSS6-2-S762A-V5) or proprotein convertase 7 (PC7-V5). Expression was detected in the cell lysate (CL) and cell media (CM) (n = 3). (TIF)</p

    Identification of TMPRSS6 isoforms interacting partners.

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    A) BioVenn overlap of TMPRSS6 isoforms 2, 3 and 4 interacting partners (fold change vs mock ≥ 3) identified by mass spectrometry analysis of TMPRSS6 V5-tagged immunoprecipitation from transfected Hep3B cell membrane preparations. B) STRING interactome of 49 common protein partners.</p

    TMPRSS6 cleaves TfR1.

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    A) TfR1-HA was transfected with one or two TMPRSS6-V5 tagged isoforms. Expression was detected in the cell lysate (CL) and cell media (CM) (n = 3). B) TfR1 cleavage quantification. Results are presented as TFR1 cleavage (%) relative to TFR1 cleavage by TMPRSS6 isoform 2. Statistical significance was assessed using one sample T test, *p < 0.02. The means ± SD are presented (n = 3).</p

    Design and Synthesis of Potent, Selective Inhibitors of Matriptase

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    Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1–P4 substrate recognition sequence of the enzyme. Preliminary structure–activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor <b>1</b> and matriptase

    Known protein substrates obtained from MEROPS [32] and UniProt [66] showing the P4-P4′ cleavage specificity in natural substrates of matriptase, matriptase-2, matriptase-3, HAT, hepsin and corin where cleaved as native proteins <i>versus</i> denatured peptides in PICS.

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    <p>Known protein substrates obtained from MEROPS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Rawlings1" target="_blank">[32]</a> and UniProt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Magrane1" target="_blank">[66]</a> showing the P4-P4′ cleavage specificity in natural substrates of matriptase, matriptase-2, matriptase-3, HAT, hepsin and corin where cleaved as native proteins <i>versus</i> denatured peptides in PICS.</p

    Analysis of Subpocket Selectivity and Identification of Potent Selective Inhibitors for Matriptase and Matriptase‑2

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    We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homologous proteases matriptase and matriptase-2 across subpockets using docking simulations. We observed that the farther away a subpocket is located from the catalytic site, the more pronounced its role in selectivity. As a result of our exhaustive virtual screening, we biochemically validated novel potent and selective inhibitors of both enzymes

    Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries - Figure 4

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    <p><b>A. <i>Upper panel</i>.</b> MALDI-TOF spectrum of the synthetic peptide AVIGRKFGDP. The sample contained a minor synthesis contaminant of AVIGRKFGD. The experimental determined [M+H]<sup>+</sup> is 1059.52 Da (predicted m/z is 1059.22 Da). The sequence is presented by the spectra. The two asterix peaks represent the MALDI matrix peaks which are found in all the spectra in this m/z range. <b><i>Second panel.</i></b> MALDI-TOF spectrum of matriptase-2. <b><i>Third panel.</i></b> MALDI-TOF spectrum of the synthetic peptide added to matriptase-2 at 0 h. <b><i>Lower panel.</i></b> MALDI-TOF spectrum of the assay reaction products generated after incubation of the synthetic peptide with matriptase-2 for 18 h. The spectral peak at 1059.52 m/z disappeared and a new peak at 515.31 Da appeared corresponding to the cleavage product [AVIGR+H]<sup>+</sup> (predicted m/z is 514.96 Da). <b>B. </b><b><i>Upper panel.</i></b> MALDI-TOF spectrum of the dimethylated (dm) synthetic peptide (dm)AVIGR(dm)KFGDP. The experimental determined [M+H]<sup>+</sup> is 1115.52 Da. The sequence is presented on the spectral peak. The two asterix peaks represent the MALDI matrix peaks and they can be found in all the spectra. <b><i>Second panel.</i></b> MALDI-TOF spectrum of matriptase-2. <b><i>Third panel.</i></b> MALDI-TOF spectrum of the reaction products after incubation of the dimethylated synthetic peptide with matriptase-2 added at 0 h. <b><i>Lower panel.</i></b> MALDI-TOF spectrum of the reaction products after incubation of the dimethylated synthetic peptide with matriptase-2 for 18 h. The peak at 1115.52 Da disappeared and a new peak at 543.30 Da appeared, corresponding to [dAVIGR+H]<sup>+</sup>. Red (dm) is dimethylation.</p
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