“<i>In vitro</i>” cleavage of purified ACT by calpain.

<p>Western blot analysis of an <i>in vitro</i> proteolysis assay in which purified ACT (5 µM) and calpain (100∶1 mol ratio) were incubated for 10 minutes at room temperature. Reactions were terminated by boiling the samples for 10 min in SDS-PAGE buffer. When indicated, calmodulin (10 µM) or calpeptin (10 µM) were included in the incubation buffer. MAb 3D1 was used to detect the N-terminus domain of the toxing after m-calpain cleavage. Results from one representative experiment out of three performed are shown in the figure (A). MAb 9D4 was used to detect the RTX domain of the toxin after m-calpain cleavage. Results from one representative experiment out of three performed are shown in the figure (B). The sample resulting from the proteolytic assay in the presence of calmodulin (corresponding to lane 2, from left to right, in the Western blot shown in panel A) was separated by molecular exclusion chromatography. The different fractions collected were analyzed by SDS-PAGE (8.5% polyacrylamide) and Western blot stained with the anti-AC monoclonal antibody MAb 3D1 (C). Catalytic activity of the intact purified ACT (1 nM) and of the fraction containing the two purified peptide bands (1 nM, final concentration with 2 nM calmodulin) was determined by ELISA as described in Methods section (D).</p

Upon cleavage by calpain, the “soluble” AC domain migrates to different subcellular organelles.

<p>Subcellular localization of the “soluble” AC domain as determined by confocal microscopy. Incubation of J774A.1 cells with ACT (35 nM) results in a colocalization of the “soluble” AC domain with the nucleus and the mitochodria after 10 min. Migration is prevented by pretreatment of cells with calpeptin (100 µM). Cells were preincubated for 30 min with the inhibitor prior to ACT addition, then incubated for 10 min with the toxin, washed three times, fixed and permeabilized to analyze AC domain by immunohistochemistry (A). Z-stack images obtained in the same conditions as in panel A, showing colocalization of “soluble” AC domain with the nucleus and mitochondria, and prevention of migration by pretreatment of cells with calpeptin (100 µM). Anti-nucleoporin p62 staining shows no colocalization of “soluble” AC domain with the nuclear envelope (B). Detection of the “soluble” AC domain into isolated nuclei, mitochondria or ER by Western blot analysis. Nuclear, ER and mitochondrial fractions isolated from cells treated with ACT (35 nM) for 10 min were analyzed by Western blot and stained with the anti-AC MAb 3D1. Anti-histone H2B antibody and anti-VDAC/porin antibody were used to stain histone H2B and the VDAC/porin as molecular markers of the nuclei and mitochondria, respectively. Calregulin was used as marker for ER fractions (C). Adenylate cyclase activity is enhanced very significantly both in nuclear extracts and mitochondrial fractions obtained from 35 nM ACT treated cells. cAMP production was determined as described in <i>Materials and Methods</i> (D).</p

Fluorimetric calpain activity assay.

<p>Fluorimetric calpain activity assay using a cell permeable FRET-based substrate (DABCYL)-TPLK-SPPPSPRE(EDANS)RRRRRRR-NH2 (20 µM) incorporated into J774A.1 cells. Kinetics of the fluorescence signal of the fluorogenic substrate, recorded in control cells and cells treated with ACT (5 and 35 nM). The inset shows the toxin concentration dependence of the substrate fluorescence signal intensity. Fluorescence intensity values (IF) taken after 30 min cell incubation with the corresponding toxin amount are plotted. Data are the average ±SD value obtained from three independent experiments (A). Effect of cysteine protease inhibitors (100 µM calpeptin, 0.5 mM NEM and 10 µM α-iodoacetamide) on the proteolysis of the fluorogenic FRET-substrate in ACT-treated cells (B).</p

Calpain, the cellular cysteine protease responsible for ACT cleavage into J774A.1 cells.

<p>Effect of several inhibitors of cysteine proteases (100 µM calpeptin, 0.5 mM NEM and 78 nM SJA6017) on the proteolytic processing of ACT (35 nM) in macrophages, upon 10 minutes incubation of cells with the toxin, as assayed by Western blot of a cytosolic fraction purified from the ACT-treated cells. Cells were previously incubated with the various inhibitors for 30 min, before toxin addition (A) Densitometric quantification of the two major bands detected in the immunoblots (B). Levels of significance were determined by a two-tailed Student's t-test, and a confidence level of greater than 95% (p<0.05) was used to establish statistical significance. *p<0.001 with respect to 50 kDa band; #p<0.001 with respect to 45 kDa band.</p

Effect of an activator and an inhibitor of soluble adenylate cyclases (sAC) on the enzyme activity of <i>in vitro</i> cleavage N-terminal fragments of ACT.

<p>The catalytic activity of N-terminal fragments purified from an <i>in vitro</i> ACT-cleavage assay was measured in presence of HCO₃^- (activator of sAC) (A) and KH7 (inhibitor of sAC) (B) at two different concentrations. The cyclase activity was determined as described in <i>Materials and Methods</i>. *p<0,05; ***p<0,001 with respect to N-terminal fragments in the absence of the compounds.</p

ACT catalytic domain translocation and cAMP production in membranes and cytosol in the presence of calpain inhibitors.

<p>J774A.1 cells were incubated for 10 min with 35 nM ACT in the presence or absence of calpain inhibitors (100 µM calpeptin and 78 nM SJA6017), translocation efficiency (A) and cAMP production in membrane and cytosolic fractions were determined as described in <i>Materials and Methods</i> (B). ***p<0.001 with respect to ACT in the absence of inhibitors.</p

ACT is proteolytically cleaved in J774A.1 cells.

<p>Western blot analysis of cytosolic extracts (A) and membrane fractions (B) purified, as described in <i>Materials and Methods</i>, from J774A.1 cells treated with ACT (35 nM) at different incubation times (0–30 min) or different toxin concentrations (0–35 nM), and stained with anti-AC monoclonal antibody MAb 3D1 for the cytosolic fraction, and with MAb 9D4 for the membrane fraction. (C) Time course of the 200 and 150 kDa bands in membrane fractions of cells treated with 5 nM ACT at different incubation times. J774A.1 cells were incubated with ACT at 4°C and non-bound ACT was washed in ice-cold PBS as described in <i>Materials and Methods</i>. Then, cells were challenged at 37°C and the full length toxin and the 150 kDa fragment were determined in the membrane fractions of the cells with MAb 9D4 (left-hand panel) and the full length toxin was also detected by MAb 3D1 (right-hand panel). Densitometric quantification of the bands detected in the immunoblots is shown below the Western blots. A representative experiment from three independently performed assays is shown in (A) and (B), and a representative experiment from two independently performed assays is shown in (C).</p

Activation of the m-calpain isoform in the ACT-treated cells.

<p>Western blot of the cytosolic fraction isolated from J774A.1 cells treated with ACT (35 nM) or from cells transfected with anti m-calpain siRNA and treated with ACT (35 nM) (A). Kinetics of the proteolytic cleavage of the FRET-based calpain specific substrate for ACT-treated cells (35 nM toxin), and cells in which m-calpain expression has been silenced by transfection with the corresponding siRNA, or mock cells transfected with scrambled control siRNA, or cells pre-incubated with SAJ6017 (78 nM), a specific m-calpain inhibitor. The figure is a representative experiment from three similar assays performed under identical conditions (B). Assay of calpain activity using FITC-labelled α-casein as substrate. FITC-α-casein hydrolysis by calpain is determined from in J774A.1 cell extracts incubated with FITC-labelled α-casein in the presence or absence of 1 mM CaCl₂. Protease activity was quantified in 10% polyacrylamide gel by determining fluorescence intensity of the non-hydrolysed FITC-α-casein band. Non-digested FITC-α-casein and cell extracts without calcium were used as negative controls (C).</p

Protein and mRNA expression of wt LDLR and LDLR variants in CHO-<i>ldlA7</i> transfected cells.

<p>Cells were transfected with the corresponding plasmids carrying the mutations of interest, LDLR was overexpressed for 48 h and then (A) protein expression was analyzed by Western blot, (B) the relative band intensities of both mature and precursor LDLR forms were calculated as the ratio of 160 kDa or 130 kDa LDLR band intensity to that of GAPDH (C) relative LDLR mRNA expression determined by qRT-PCR (normalised to GAPDH). A representative experiment from three independently performed assays is shown in (A). The values in (B) and (C) represent the mean of triplicate determinations (n = 3) and (n = 2), respectively; error bars represent ±SD. Levels of significance were determined by a two-tailed Student’s t-test, and a confidence level of greater than 95% (p<0.05) was used to establish statistical significance. *p<0.001 compared to the LDLR wt 160 kDa band and #p<0.001 compared to LDLR wt 130 kDa band using a Student’s t-test.</p

Ca²⁺ Influx and Tyrosine Kinases Trigger <i>Bordetella</i> Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

<div><p>Humans infected with <i>Bordetella pertussis</i>, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca²⁺ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.</p></div