Journal: Nature Communications

Article Title: Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection

doi: 10.1038/s41467-021-21972-0

Figure Lengend Snippet: a Protein–protein docking analysis of a homology model for the TMPRSS2 extracellular fragment (green, PDB 1z8g) and α 1 AT (gray, PDB 3cwm) and computationally calculated binding free energy (ΔG calc ) of the complex. b Detailed view on α 1 AT-TMPRSS2 binding interface. The sidechains of α 1 AT (gray) residues are represented with sticks, while sidechains of TMPRSS2 (green) are shown with balls and sticks. Hydrogen atoms are omitted for clarity, carbon, oxygen, nitrogen or sulfur atoms of amino acid side chains depicted in light blue, red, dark blue or yellow, respectively. The hydrophobic patch near the TMPRSS2 catalytic triad is highlighted with a green transparent surface. c α 1 AT inhibits cell-associated TMPRSS2 activity. HEK293T cells were transfected with a TMPRSS2 expression plasmid and treated with Prolastin (α 1 AT, blue), camostat mesylate (CM, gray) or E-64d (green) followed by incubation with the fluorogenic TMRPSS2 protease substrate BOC-Gln-Ala-Arg-AMC. Graph shows the relative area under the curve analysis of fluorescence intensities over 2 h that were corrected by values for mock-transfected HEK293T cells. d α 1 AT inhibits recombinant TMPRSS2 enzyme activity. Recombinant human TMPRSS2 was mixed with Prolastin (α 1 AT, blue) or CM (gray) prior to addition of fluorogenic TMPRSS2 protease substrate BOC-Gln-Ala-Arg-AMC, graph shows relative fluorescence intensities after 3 h of incubation. The mean ± SEM of n = 2 ( c ) or n = 3 ( d ) independent experiments in biological duplicates are shown (ordinary one-way ANOVA with Dunett´s multiple comparison test). Source data are provided as a Source data file.

Article Snippet: For assessing the activity of recombinant human TMPRSS2, 25 µl of serially diluted Prolastin or inhibitor control in assay buffer (50 mM Tris-HCL, 0.154 mM NaCl pH 8.0) were incubated with 25 µl of recombinant TMPRSS2 enzyme for 10 min at 37 °C, followed by addition of 20 µM BOC-Gln-Ala-Arg-AMC protease substrate.

Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Fluorescence, Recombinant

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: tpp1 − cells show reduced proteolytic activity. Cell lysates from equivalent numbers of 16-hour starved cells were incubated with 50 μM AAF-AMC, and the fluorescence of the released fluorophore AMC was measured 30 minutes after the addition of substrate. The fluorescence was normalized to that of wild type for each independent experiment. Values are means±SEM, n =4. ** P <0.01; ns, not significant (one-sample t -test with comparison to the value 100%). hsTpp1, human TPP1.

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Activity Assay, Incubation, Fluorescence, Comparison

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: Dictyostelium and human TPP1 localize to the lysosome in Dictyostelium cells. Cells were starved for 16 hours in shaking culture, stained with Lysotracker, and then imaged in chamber slides by confocal microscopy; images are representative of three independent experiments. hsTpp1-GFP, human TPP1 fused to GFP. Scale bar: 10 μm.

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Staining, Confocal Microscopy

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: Tpp1 affects the development of spores. (A) Ten million cells were allowed to develop on filter pads for 24 hours, and then spores were collected and counted. (B) Equivalent numbers of the collected spores were treated with detergent and plated onto a lawn of K. aerogenes bacteria, and the number of resultant colonies was counted. Values are means±SEM, n =4. * P <0.05 (one-way ANOVA, Tukey’s test).

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Bacteria

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: tpp1 − cells show increased intracellular autofluorescent material. (A) Cells were starved for 16 hours in chamber slides, and intracellular autofluorescent material in cells at the periphery of cell aggregates was imaged by using confocal microscopy; images are representative of three independent experiments. Scale bar: 20 μm. (B) Cells were starved in shaking culture for 16 hours, and the cellular autofluorescence was assayed by using flow cytometry. The average fluorescence per cell was normalized to that of wild type. Values are means±SEM, n =3. * P <0.05; ns, indicates not significant (one-sample t -test with comparison to the value 100%). (C) Cells growing on a bacterial lawn were collected and assayed as described in B. Values are means±SEM, n =3. The differences between wild-type and either tpp1 − or tpp1 − /cotB::ddTpp1 are not significant (one-sample t -test with comparison to the value 100%).

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Confocal Microscopy, Flow Cytometry, Fluorescence, Comparison

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: Tpp1 affects the autophagic response of cells. (A) The size of cells cultured continuously in autophagy-stimulating media (ASM) was measured daily by flow cytometry using forward scatter. Values are means±SEM, n =5. * indicates that differences between wild type and tpp1 − were significant with P <0.05, ** P <0.01 and *** P <0.001 (one-way ANOVA, Tukey’s test). Differences between wild type and tpp1 − /cotB::ddTpp1 were not significant for any time point. (B) Every 2 days, an aliquot was taken from a culture of cells continually cultured in ASM and then spread onto a lawn of K. aerogenes bacteria, and the viability of cells was measured by counting the resulting colonies. Values are means±SEM, n =4. * indicates that differences between wild type and tpp1 − were significant with P <0.05 (one-way matched values ANOVA, Tukey’s test). Differences between wild type and tpp1 − /cotB::ddTpp1 were not significant for any time point. (C) Cells cultured for 8 days in ASM were allowed to settle in a glass chamber slide, and then cells were imaged with an inverted phase-contrast microscope; images are representative of three independent experiments. Scale bar: 20 μm.

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Cell Culture, Flow Cytometry, Bacteria, Microscopy

Journal: Disease Models & Mechanisms

Article Title: Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

doi: 10.1242/dmm.018820

Figure Lengend Snippet: Development of tpp1 − cells is inhibited in the presence of chloroquine or ammonium chloride. (A) Clonal cell colonies were grown on SM/5 plates supplemented with 3 mM chloroquine and then spread with a lawn of K. aerogenes bacteria; images were taken after 8 days and are representative of three independent experiments. (B) Cells were starved in a 24-well plate for 14 hours in PBM (for PBM buffer components, see Materials and Methods), PBM with 5 mM chloroquine, or PBM with 20 mM ammonium chloride. All images are representative of three independent experiments. Scale bars: 1 mm (A); 200 μm (B).

Article Snippet: A solution of 100 μM TPP1 substrate AAF-AMC HCL (Bachem) was made in 100 mM sodium acetate pH 3.8, and 50 μl of cell lysate (or, as a control, 50 μl of buffer) was mixed with 50 μl of AAP-AMC solution in a 96-well opaque plate (Nunc) to give a final concentration of 50 μM Ala-Ala-Phe-AMC.

Techniques: Bacteria