Tatiana El Hage 1,2,Séverine Lorin 3, Paulette Decottignies 4,5, Mojgan Djavaheri-Mergny 6 and François Authier 1,2

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1 Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP-ribosylating and apoptotic effects Tatiana El Hage 1,2,Séverine Lorin 3, Paulette Decottignies 4,5, Mojgan Djavaheri-Mergny 6 and François Authier 1,2 1 INSERM, Châtenay-Malabry, France 2 Université Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France 3 JE 2493, Université Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France 4 CNRS, UMR 8619, Orsay, France 5 Université Paris-Sud, Orsay, France 6 INSERM VINCO U916, Institut Bergonié, Bordeaux, France Keywords cathepsin; endocytosis; endosome; Pseudomonas exotoxin A; translocation Correspondence F. Authier, INSERM, Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, Châtenay-Malabry, France Fax: Tel: francois.authier@u-psud.fr (Received 21 March 2010, revised 4 June 2010, accepted 12 July 2010) doi: /j x To assess Pseudomonas exotoxin A () compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized with the use of the in vivo rodent liver model following toxin administration, cell-free hepatic endosomes, and pure in vitro protease assays. taken up into rat liver in vivo was rapidly associated with plasma membranes (5 30 min), internalized within endosomes (15 60 min), and later translocated into the cytosolic compartment (30 90 min). Coincident with endocytosis of intact, in vivo association of the catalytic -A subunit and low molecular mass -A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the -degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of -A. Cell-free endosomes preloaded in vivo with intraluminally processed and extraluminally released intact and -A in vitro in a ph-dependent and ATP-dependent manner. Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of infection, as assessed by the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activation, and DNA fragmentation. In an in vitro assay, intact induced ADP-ribosylation of EF-2 and mitochondrial release of cytochrome c, with the former effect being efficiently increased by a cathepsin B cathepsin D pretreatment. The data show a novel processing pathway for internalized, involving cathepsins B and D, resulting in the production of fragments that may participate in cytotoxicity and mitochondrial dysfunction. Abbreviations DT, diphtheria toxin; EEA1, early endosome antigen-1; EF-2, elongation factor-2; ER, endoplasmic reticulum;, exotoxin A; HA, hexa-d-arginine; LRP1, low-density lipoprotein receptor-related protein 1; PA, pepstatin-a; SD, standard deviation; a 2 MG, a 2 -macroglobulin. FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3735

2 Proteolysis of in rat hepatic endosomes T. El Hage et al. Introduction Exotoxin A () is considered to be the most toxic factor secreted by Pseudomonas aeruginosa, a Gramnegative opportunistic pathogen infecting immunocompromised individuals and burn victims [1]. is a 613 amino acid A B exotoxin that kills cells by inhibition of protein synthesis and programmed cell death [2,3]. is secreted as a single polypeptide chain composed of three structural and functional domains: domain Ia (amino acids 1 252), which binds to the a 2 -macroglobulin (a 2 MG) low-density lipoprotein receptor-related protein 1 (LRP1) receptor present in animal cells [4]; domain II (amino acids ), which contains a furin cleavage site (Arg276-Gln277- Pro278-Arg279), the Cys265 Cys287 disulfide bond, and a protein translocating sequence (amino acids ) [5,6]; and domain III (amino acids ), which contains the ADP-ribosylating enzyme [2]. To access and ADP-ribosylate its cellular target, elongation factor-2 (EF-2), must be transported across the cellular membrane and into the cytoplasm. This is initiated by cell surface binding of to the a 2 MG LRP1 receptor [4], which is followed by internalization of the toxin receptor complex to the endosomal apparatus by clathrin-dependent and clathrin-independent mechanisms [7]. Two subcellular compartments have been proposed as being physiologically relevant to the mechanism of translocation of internalized into the cytosol. The first translocation pathway has been proposed to operate at an early stage of endocytosis from endocytic vesicles [8,9]. Thus, significant translocation of across the endosomal membrane of mouse lymphocytes was demonstrated, and required exposure of to low endosomal ph and ATP hydrolysis [10]. Other studies have proposed that internalized can be retrogradely transported to the endoplasmic reticulum (ER) for retrotranslocation to the cytosol through the Sec61 complex [11]. The ER trafficking pathway of might have multiple routes [7], one being the previously characterized KDEL pathway involving the REDLK C-terminal sequence of the toxin [12]. Whatever the pathway enabling cytosolic delivery of, activating processes have been proposed to occur at various stages of trafficking. These activating steps include furin-mediated cleavage at the Arg279- Glu280 peptide bond [13], reduction of the disulfide bond linking Cys265 and Cys287 [14], and removal of the C-terminal Lys [15]. Thus, for full ADP-ribosylation of cytosolic EF-2, it was previously suggested that intracellular production of a 37 kda C-terminal fragment must occur by the sequential action of a furin-like protease and an undiscovered reductive enzyme [2,13,16]. These observations are consistent with the toxin-resistant phenotype of cells lacking furin, which can be abolished by transfection with a cdna encoding furin [17]. However, although proteolytic and reductive processing of should be required for cytotoxicity through the retrograde transport pathway [18], it has not been clearly determined whether requires proteolytic and or reductive processing activation to reach the cytosol through the endosomal pathways and kill cells [19]. Hence, recent studies have suggested that cytotoxicity results largely from endosomal translocation of the intact nonproteolyzed and nonreduced polypeptide toxin [19]. At present, no in vivo data exist to support a specificity of requirement for processing and reduction according to the translocation pathway used (endosome or ER). Consequently, in the present study, we used the in situ rat liver model system following toxin administration to rats and cell-free hepatic endosomes to relate the endosomal processing of internalized to toxin cytotoxicity in a physiological state. Following administration of to rats, rapid endocytosis of the intact unprocessed was observed, coincident with the endosomal association of the -A subunit (fast association) and low molecular mass -A fragments (slow association). Our results assign an important role to endosomal acidic cathepsins B and D in generating fragments displaying high in vitro ADP-ribosyltransferase activity towards cytosolic EF-2. We report on the in vivo association of and -A with cytosolic fractions, and the in vitro ATP-dependent and ph-dependent translocation of and -A from cell-free endosomes into the external milieu. Finally, the mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3 and DNA fragmentation were detected in cytosolic fractions isolated 2 h after treatment, relating for the first time activation of the intrinsic apoptotic pathway with cytotoxicity in a physiological state. Results In vivo endocytosis and metabolic fate of in rat liver The kinetics of in vivo uptake of at the hepatic cell surface (plasma membranes) (Fig. 1A) and intracellularly (endosomes) (Fig. 1B) were assessed first. Rats were given an intravenous injection of native 3736 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

3 T. El Hage et al. Proteolysis of in rat hepatic endosomes A Nonreducing conditions -A Plasma membranes B (min, postinjection) Nonreducing conditions -A Endosomes (min, postinjection) Reducing conditions (66 kda) -A Reducing conditions (66 kda) -A kda 15 kda Fig. 1. Kinetics of appearance of in hepatic plasma membranes and endosomes after toxin administration. Rat hepatic plasma membrane (A) and endosomal fractions (B) were isolated at the indicated times after the in vivo administration of native, and evaluated for their content of internalized toxin by nonreducing (upper blots) and reducing SDS PAGE (lower blots) followed by western blot analysis with the polyclonal antibody against. Each lane contained 10 lg (plasma membranes) or 30 lg (endosomes) of protein. The arrows to the left of each panel indicate the mobilities of intact ( 66 kda), -A ( 37 kda), and unknown degradation fragments. Molecular mass markers are indicated to the right of the reducing blots. The antibody against also binds to undefined plasma membrane proteins distinct from under nonreducing conditions [upper blot in (A)] in both control and toxin-injected rats, one of which had a molecular mass identical to that of -A. (15 lg per 100 g body weight) and killed 5 90 min postinjection. Following preparation of hepatic subcellular fractions, the amount of internalized was determined by SDS PAGE followed by western blot analyses with antibody directed against -A. It was assumed that the in vivo generation of free -A was attributable to both reductive and proteolytic cleavages occurring within the sequence. Thus, both processing pathways were analyzed, under either nonreducing (cleavage analysis at the Cys265 Cys287 disulfide bond; upper blots in Fig. 1) or reducing (cleavage analysis at peptide bonds; lower blots in Fig. 1) conditions. association with plasma membranes was rapid (5 min postinjection) and maximal at 5 30 min postinjection, before decreasing with time (Fig. 1A). A transient association of -A with plasma membranes was also observed under reducing and nonreducing conditions at min postinjection (Fig. 1A). As compared with plasma membranes, endosomal association of both and -A was slightly delayed, with the maximum being observed at min () or min (-A) (Fig. 1B). Low molecular mass fragments (< 25 kda) were immunodetected, especially in endosomal fractions under reducing conditions (Fig. 1B, lower blot). Although it has been suggested that it is the receptor complex that is internalized into toxintreated cells, there are no published reports on the fate of the receptor during toxin endocytosis. To determine whether the receptor was cointernalized along with the toxin, the in vivo effect of treatment on the a 2 MG LRP1 receptor in the rat liver endosomal fraction was determined by immunoblotting (Fig. 2A, upper blot). A high concentration of a membrane-bound 80 kda fragment of LRP1 containing the tail epitope was found in the endosomal fraction isolated from control rats. The extensive fragmentation of LRP1 within hepatic endosomes may explain, in part, the failure to detect intact LRP1 by us (this study) and others [20]. In vivo injection of native effected a rapid increase in endosomal truncated LRP1, with maximal accumulation at 5 15 min postinjection. By 60 min postinjection, the 80 kda LRP1 species had returned to basal levels (Fig. 2A, upper blot). However, the level of the endosomal marker early endosome antigen-1 (EEA1) was not modified after treatment (Fig. 2A, lower blot). LRP1 enables endocytosis of and various other ligands among such as a 2 MG [21]. To examine the effect of a 2 MG on the uptake of into hepatic endosomes, a 2 MG (15 lg per 100 g body weight) was coinjected with into rats (Fig. 2B). Endosomal association of intact and -A was reduced by a 2 MG coinjection. We have previously reported that antibodies reacting with the ER-retention KDEL motif are useful in assessing the integrity of the C-terminal region of cholera toxin [22]. As it was unknown whether antibodies FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3737

4 Proteolysis of in rat hepatic endosomes T. El Hage et al. A α-lrp1 (tail) B Arbitrary units (66 kda) -A C α-eea (min, postinjection) Furin Dithiothreitol (66 kda) -A * * LRP1 (80 kda) (min, postinjection) (min, postinjection) EEA1 (180 kda) /a 2 MG (min, postinjection) kda (min, postinjection) -A Fig. 2. Characterization of endocytosis into the endosomal apparatus. (A) Changes in LRP1 concentration in the endosomal fraction following injection into rats. Hepatic endosomal fractions were isolated at the indicated times after the in vivo administration of native, and evaluated for their content of LRP1 (upper blot) or EEA1 (lower blot) by reducing SDS PAGE followed by western blot analysis. Each lane contained 30 lg (a-lrp1 blot) or 50 lg (a-eea1 blot) of endosomal protein. The LRP1 bands were quantified by scanning densitometry, and the signal intensities for the -treated rats were expressed as a percentage (mean ± SD) of the signal intensity for the control rats (lane )). *P < 0.05 for the differences between 5 min or 15 min and control rats ()). The arrows to the right indicate the mobilities of membranebound LRP1 fragment ( 80 kda) or EEA1 ( 180 kda). Uncleaved LRP1 ( 600 kda) was not observed in endosomal fractions from control and toxin-injected rats. (B) Effect of a 2 MG treatment on the internalization of. Rat hepatic endosomal fractions were isolated at the indicated times after the in vivo coadministration of and a 2 MG (15 lg per 100 g body weight), and evaluated for their content of internalized toxin by reducing SDS PAGE followed by western blot analysis with the polyclonal antibody against. Each lane contained 50 lg of endosomal protein. The arrows to the left indicate the mobilities of intact ( 66 kda), -A ( 37 kda), and unknown degradation fragments. Molecular mass markers are indicated to the right. (C) Assessment of immunoreactivity of antibody against KDEL for native and internalized. was either untreated (left blot, lane )) or digested in vitro with 100 UÆmL )1 Æmg )1 furin and 10 mm dithiothreitol (middle blot, lane +), and samples were then analyzed by reducing SDS PAGE followed by western blotting with polyclonal antiserum against the synthetic peptide KX 5 KDEL. -A was detected under the latter experimental conditions. Rat liver endosomal fractions were then isolated at the indicated times after the in vivo administration of, and evaluated by western blotting for their immunoreactivity with polyclonal antibody against KX 5 KDEL (blot on the right) [22]. The antibody against KX 5 KDEL also binds to undefined endosomal proteins distinct from, both in control and in toxin-injected rats, whose levels have been shown to be modified by toxin treatment [22]. Each lane contained 80 lg of endosomal protein. The mobilities of intact ( 66 kda) and -A ( 37 kda) are indicated. α-kx 5 KDEL α-kx 5 KDEL against KDEL bind to the C-terminal sequence REDLK (which resembles the ER motif KDEL), we first characterized antibodies against KDEL for their binding to native and -A by western blot analysis (Fig. 2C, left and middle blots). One antibody, anti-kx 5 KDEL, bound to -A but not to native (Fig. 2C, left and middle blots), whereas the others, anti-ksekkdel and anti-kavkkdel, did not show any immunoreactivity (results not shown). Therefore, the antibody against KX 5 KDEL was used to assess the integrity of the REDLK peptide in endosomal -A under reducing conditions (Fig. 2C, right blot). KDEL immunoreactivity to internalized -A was detected in endosomal fractions isolated from the livers of rats killed at min postinjection, with kinetics similar to those of -A uptake into endocytic components (Fig. 1B), suggesting that the C-terminal motif REDLK might not be completely removed from -A within the endosomal apparatus. Endosomal proteolysis of internalized by cathepsins B and D. To confirm the endosomal proteolysis of internalized under conditions that maintained endosome integrity, we used cell-free endosomes containing in vivo internalized (Fig. 3A,B). Endosomes were isolated 30 min following injection, and intact endocytic vesicles were incubated for various times at 3738 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

5 T. El Hage et al. Proteolysis of in rat hepatic endosomes A B kda Arbitrary units (66 kda) -A ph 7 ph 7 + ATP (min) PMSF -A subunit (min) (ATP) ph 7 + ATP (Inhibitor) (min) PA EDTA HA (66 kda) -A E64 Fig. 3. Assessment of -degrading activity associated with hepatic endosomes. (A) Rat hepatic endosomal fractions were isolated 30 min after administration (15 lg per 100 g body weight) and incubated for the indicated times at 37 C in isotonic buffer containing 0.15 M KCl, 25 mm Hepes (ph 7), 5 mm MgCl 2, and 6 mm CaCl 2, in the presence or absence of 10 mm ATP. The integrity of was then evaluated by reducing SDS PAGE followed by western blotting with the polyclonal antibody against. Each lane contained 50 lg of endosomal protein. The arrows to the right indicate the mobilities of intact ( 66 kda), -A ( 37 kda), and unknown degradation fragments. Molecular mass markers are indicated to the left. and -A signals were quantified by scanning densitometry, and the signal intensities after a 30 min incubation were expressed as a percentage (mean ± SD) of initial values (0 min) (lower panel). (B) Rat hepatic endosomal fractions were isolated 30 min after administration (15 lg per 100 g body weight) and incubated at 37 C in isotonic buffer containing 0.15 M KCl, 25 mm Hepes (ph 7), 5 mm MgCl 2,6mM CaCl 2 and 10 mm ATP for the indicated times in the presence or absence (lane )) of 2 mm phenylmethanesulfonyl fluoride (PMSF), 10 lgæml )1 PA, 5 mm EDTA, 10 lm HA, or 10 lm E64. The integrity of was then evaluated by reducing SDS PAGE followed by western blotting with the polyclonal antibody against. Each lane contained 50 lg of endosomal protein. The arrows to the left and right indicate the mobilities of intact ( 66 kda), -A ( 37 kda), and unknown degradation fragments. (C) Native (10 lg) was incubated at 37 C with cathepsin D (Cath-D) or cathepsin B (Cath-B) (5 UÆmL )1 Æmg )1 ) in 50 mm citrate phosphate buffer (ph 4 6) or 50 mm Hepes (ph 7) in the presence of 10 mm CaCl 2 and 10 mm dithiothreitol for the indicated times. The integrity of was then evaluated by reducing SDS PAGE followed by western blotting with the polyclonal antibody against. The arrows to the left and right indicate the mobilities of intact ( 66 kda), -A ( 37 kda), and unknown degradation fragments. C + Cath-D + Cath-B ph (min) (66 kda) -A neutral ph (ph 7) and 37 C in an isotonic buffer (which mimicked the intracellular milieu) in the presence or absence of ATP, the substrate of the vacuolar H + -ATPase pump (Fig. 3A). Immunoblot analyses showed a progressive loss of intact and -A in the presence of ATP, with concomitant generation of and -A fragments. Incubation in the absence of ATP revealed a small amount of degradation for intact, whereas no degradation was observed for -A (Fig. 3A). We next examined the effects of various protease inhibitors on the proteolysis of endosomal and -A, using cell-free endosomes preloaded with toxin in vivo and incubated in vitro at ph 7 in the presence of ATP (Fig. 3B). Western blot analysis with the antibody against revealed that the endosomal -degrading activity was partially inhibited by the aspartic acid protease inhibitor pepstatin-a (PA), the cysteine protease inhibitor E64, and the metalloprotease inhibitor EDTA. The inhibition of -degrading activity by PA and E64, its low ph optimum and its presence in the endosomal lumen as a soluble form (results not shown) suggested cathepsins B and D as likely candidates for this activity. We therefore examined the hydrolysis of by pure cathepsins B and D at ph 4 7 (Fig. 3C). FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3739

6 Proteolysis of in rat hepatic endosomes T. El Hage et al. A (66 kda) kda Cathepsin B Cathepsin D ph of medium 4a 6a 4b 6c 6d 4d 6b B -B -A 15 4c Incubation time (h) 1AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDN ALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGN QLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRL HFPEGGSLAALTAHQACHLPLETFTRHRQPR GWEQLEQCGYPVQRLVALYLAARLSWNQVDQV IRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCP VAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYV FVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGA LLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWP LAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK 613 Fig. 4. Structural characteristics of fragments generated by cathepsin B and cathepsin D. (A) Native (10 lg) was incubated with bovine cathepsin B or cathepsin D (5 UÆmL )1 Æmg )1 ), or a mixture of both, at 37 C for the indicated times in 50 mm citrate phosphate buffer (ph 4 6). The incubation mixtures were then analyzed by reducing SDS PAGE followed by Coomassie Brilliant Blue Staining. The major degradation products generated at ph 4 (peptides 4a c) or ph 6 (peptides 6a d) were subjected to N-terminal sequence analysis. (B) Sequences of -A and -B. The A and B moieties are connected by both a peptide bond (Arg279-Gly280) and a disulfide bridge (Cys265 Cys287). The peptides in red correspond to the N-terminal sequence of intermediates shown in (A): AEEAFDL for intermediates 1, 4a, 4b, 4d, 6a and 6c; CPVAAGECA for intermediates 6b and 6d; and PALA for intermediate 4c. Western blot analysis with an antibody against showed that cathepsins B and D degraded in a ph-dependent manner, with maximal degradation being observed at ph 4. The fragments generated by the pure cathepsins (especially cathepsin B at ph 4) had molecular masses very similar to those seen with the endosomal fractions. We then assessed the major proteolytic cleavages induced by cathepsin B and or D within the sequence at various ph values (Fig. 4A,B). The proteolysis of at ph 4 or 6 by cathepsin B and or D was analyzed by reducing SDS PAGE (Fig. 4A), and the cleavage sites in the major metabolites were determined by N-terminal sequence analysis (Fig. 4B). Edman degradation of intermediates 4a, 4b, 4d, 6a and 6c revealed the N-terminal sequence of (AEEAFDL), suggesting that the cleavage sites are located within the C-terminal region of the toxin. N-terminal sequence analysis of fragments 6b and 6d, generated at ph 6, revealed cleavages between Thr396 and Cys397 (as demonstrated by the CPVAAGECA sequence). For peptide 4c, generated at ph 4, N-terminal sequence analysis revealed the peptide PALA, suggesting cleavage between Asp499 and Pro500. Assessment of cytosolic translocation of internalized We next determined the presence of in cytosolic fractions prepared from -injected rats, using western blot analysis (Fig. 5A). The intact 66 kda toxin was strongly detected within cytosolic fractions at h postinjection, and lower but detectable immunoreactivity for -A was observed at 1 4 h under both reducing and nonreducing conditions. The translocation of endosomal into the extraendosomal milieu was then assessed with intact endosomes isolated 30 min after the injection of and incubated for 0 2 h in isotonic medium at 37 C in the presence or absence of ATP (Fig. 5B). Western blot analysis of the associated with sedimentable endosomes showed progressive decreases in immunoreactive and -A at acidic ph (ph 5) or at ph 7 in the presence of ATP. Concomitantly, immunoreactive (high level) and -A (low level) were progressively detected in the extraendosomal milieu, confirming the translocation of toxin across the endosomal membrane at acidic luminal ph. No ATP-dependent translocation of was observed in the presence of bafilomycin, the H + -ATPase inhibitor FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

7 T. El Hage et al. Proteolysis of in rat hepatic endosomes A B -A (66 kda) -A (66 kda) -A (66 kda) -A (66 kda) -A (66 kda) -A Cytosol (h, postinjection) Pellet Supernatant (endosomal medium) (extraendosomal medium) (incubation, h) Medium: ph 7 Medium: ph 7 + ATP Medium: ph 5 Medium: ph 7 + ATP + Bafilomycin Nonreducing conditions Reducing conditions Fig. 5. In vivo and in vitro assessment of the cytosolic translocation of endosomal. (A) Rat hepatic cytosolic fractions were isolated at the indicated times after the in vivo administration of native, and evaluated for their toxin content by nonreducing (upper blot) and reducing (lower blot) SDS PAGE followed by western blot analysis with the polyclonal antibody against. Each lane contained 30 lg of cytosolic protein. The arrows to the left indicate the mobilities of intact ( 66 kda) and -A ( 37 kda). (B) Membrane translocation of toxin peptides in cell-free rat hepatic endosomes containing in vivo internalized. The endosomal fraction was isolated 30 min after the administration of, and then resuspended in 0.15 M KCl containing 5 mm MgCl 2 and, when indicated, 50 mm Hepes (ph 7), 50 mm citrate phosphate buffer (ph 5), 10 mm ATP, and 1 lm bafilomycin. After the indicated times of incubation at 37 C, endosomes were sedimented by ultracentrifugation, and the pellets (endosome-associated material) and supernatants (extraendosomal material) were evaluated for their content of peptides by reducing SDS PAGE followed by western blotting with the polyclonal antibody against. Equivalent volumes of each subfraction (40 ll) were loaded onto each lane. The arrows to the left indicate the mobilities of intact ( 66 kda) and -A ( 37 kda). Potential role of cathepsins B and D in the cytotoxic activity of towards cytosolic targets We first examined whether, under conditions where was processed by cathepsins B and D, a corresponding change in the toxin cytotoxicity towards cytosolic EF-2 would be observed (Fig. 6A). was first partially processed by a mixture of cathepsins B and D at ph 4 or 6, and then incubated at neutral ph with cytosolic EF-2 in the presence of [ 32 P]NAD. A low level of ADP-ribosylation of EF-2 was evident after addition of untreated to the cytosolic fraction. After treatment of with cathepsins B and D, EF-2 labeling was increased, especially under acidic conditions (ph 6 > ph 5 > ph 4). However, cathepsin treatment of in the presence of protease inhibitors revealed [ 32 P]NAD-ribose incorporation into cytosolic EF-2 comparable to that observed in the absence of protease treatment. A role for mitochondria in -induced cell death has been previously shown with the use of human airway epithelial target cells [23]. Consequently, we examined cytochrome c release from cell-free mitochondria isolated from control rats and then treated with in vitro (Fig. 6B, upper blots). Cytochrome c association with intact rat liver mitochondria persisted during the incubation in isotonic medium, despite small but detectable release at 15 min. However, there was substantial release of cytochrome c into the resulting mitochondrial supernatant after the addition of native or that had been pretreated with a mixture of cathepsins B and D. No detectable release of cytochrome c was observed following treatment of mitochondria with a mixture of cathepsins B and D alone for the same incubation times (results not shown). To assess the physiological release of mitochondrial cytochrome c into the cytosol, hepatic cytosolic fractions isolated after the in vivo injection of into rats were analyzed for their cytochrome c content by immunoblot analysis (Fig. 6B, lower blots). Low but detectable immunoreactivity towards cytochrome c was observed in cytosol isolated from noninjected rats. In response to, a strong increase in cytochrome c was observed at 2 h, with the level remaining elevated up to 4 h. By contrast, administration of diphtheria toxin (DT) (a toxin that does not access the cytoplasm of rodent cells [24]) did not cause a detectable change in the level of cytochrome c in the cytoplasmic compartment. The involvement of caspases in -triggered programmed cell death was then analyzed by incubating hepatic cytosol isolated from -treated rats with fluorescent substrates specific for caspase-9, caspase-3 and caspase-8 (Fig. 6C, open bars). Caspase-9 and FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3741

8 Proteolysis of in rat hepatic endosomes T. El Hage et al. A EF-2 (105 kda) EF-2 (105 kda) Arbitrary units C (ratio of [ 32 P]EF2/EF2) Cathepsin-treated + cytosol i (ph of proteolysis) ADP-ribosylation of EF-2 2 DT 1 * * WB: α-ef i (ph of proteolysis) Caspase-9 B Intact mitochondria Disrupted mitochondria no toxin Native Cathepsin-treated (min of incubation) Cytochrome c (15 kda) DT Toxin (h, postinjection) Cytochrome c (15 kda) Cytochrome c (15 kda) Cytochrome c (15 kda) Caspase activity (fold stimulation) Caspase-3 Caspase-8 D Cell death (fold stimulation) 4 2 DT (min, postinjection) (h, postinjection) Fig. 6. Assessment of cytotoxic activity of cathepsin-treated towards cytosolic target and mitochondria. (A) Native (10 lg) was digested in vitro for 30 min at 37 C with a mixture of cathepsins B and D (5 UÆmL )1 Æmg )1 )in25mm Hepes (ph 7) or 25 mm citrate phosphate buffer (ph 4 6) containing 0.1 M dithiothreitol (DT) and, when indicated, 5 lgæml )1 PA and 1 lm E64 (lane 4 + i). The treated (1 lg) was then incubated for 15 min at 30 C with the EF-2 associated with the soluble cytosolic fraction (150 lg) in 0.1 M Hepes (ph 7.4) in the presence of 2 lm [ 32 P]NAD. Samples (20 lg) were then subjected to SDS PAGE and analyzed by autoradiography. The dried gels were exposed to X-ray film at )80 C for 1 3 days. The arrow to the left indicates the mobility of 32 P-labeled EF-2 ( 105 kda). Samples (20 lg) were also evaluated for their content of EF-2 using polyclonal antibody against EF-2. The arrow to the left indicates the mobility of EF-2 ( 105 kda). For each incubation condition, radiolabeled and nonradiolabeled EF-2 signal intensities were quantified by scanning densitometry, and the ratio of [ 32 P]EF-2 signal nonradiolabeled EF-2 signal was expressed as a percentage (mean ± SD) of that of untreated (lane ), 100%) (lower panel). *P < 0.05 for the differences between ph 5 or ph 4 and untreated cytosol. (B) Upper blots: rat liver mitochondria (7.5 mgæml )1 ) were incubated in either 0.15 M KCl isotonic buffer (intact mitochondria, blot at the top) or hypotonic buffer (disrupted mitochondria, lower blot) in the presence or absence of native or cathepsin-treated. After the indicated times, samples were centrifuged and mitochondrial supernatants were carefully separated and mixed with sample buffer. Equivalent volumes (30 ll) were subjected to reducing SDS PAGE followed by western blot analysis for the in vitro release of cytochrome c. The arrows to the right indicate the mobility of cytochrome c ( 15 kda). Lower blots: rat hepatic cytosolic fractions were isolated at the indicated times after the in vivo administration of native or diphtheria toxin (DT), and evaluated by western blotting with monoclonal antibody for their content of cytochrome c. Each lane contained 30 lg of cytosolic protein. The arrows to the right indicate the mobility of cytosolic cytochrome c ( 15 kda). (C) Hepatic cytosolic fractions isolated from -injected or DT-injected rats were incubated with fluorescent substrates specific for caspase-9, caspase-3, and caspase-8. The results are expressed as fold stimulation of fluorescence intensity, normalized to that seen in the control rats, and represent the mean ± SD of three determinations performed on the cytosolic fraction prepared from separate liver fractionations. (D) Histone-associated DNA fragments associated with hepatic cytosolic fractions isolated from -injected and DT-injected rats were analyzed by immunoassay. Results are expressed as fold stimulation, normalized to that seen in the control rats, and represent the mean of two determinations performed on the cytosolic fractions prepared from separate liver fractionations FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

9 T. El Hage et al. Proteolysis of in rat hepatic endosomes caspase-3 activity increased in rat liver cytosol h after the injection of, with a maximal effect of 2.7-fold (caspase-9) or 4.0-fold (caspase-3) at 4 h. No activation of caspase-8 (involved in the extrinsic death receptor pathway) was observed. No increase in caspase activity was observed in hepatic cytosolic fractions isolated from DT-injected rats (Fig. 6C, closed bars). Finally, the kinetics and extent of production of histone-associated DNA fragments in hepatic cytosolic fractions following administration into rats paralleled caspase-9 and caspase-3 activation, with DNA fragmentation being observed 2 h after injection and remaining elevated up to 4 h (Fig. 6D, open bars). No DNA fragmentation was observed in hepatic cytosolic fractions isolated from DT-injected rats (Fig. 6D, closed bars). Discussion Using the in situ liver model system, we have previously shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin D [22,25]. Using a similar methodology, others have previously shown that the plant toxin ricin follows a similar intraendosomal processing pathway, requiring ATP-dependent endosomal acidification [26]. We have recently extended these observations to DT, and demonstrated the endosomal processing of the internalized toxin in a sequential degradation pathway beginning early, prior to organelle acidification via a neutral furin activity, and followed later under acidic conditions via cathepsin D [24]. In the present work, we have evaluated the relationship between the endosomal processes and cytotoxicity of, another A B toxin functionally related to DT that has an identical intracellular target (cytosolic EF-2) [6]. Our data clearly show that internalized is susceptible to hydrolysis by cathepsins B and D, which are present in hepatic endosomes and operate at acidic ph. Comparable to the endosomal degradation of internalized CT [22,25] and ricin [26] in rat hepatic endosomes, the endosomal processing of internalized occurred mainly (if not totally) following ATP-dependent acidification of the endosomal lumen. Cytosolic translocation of endosomal was established through the immunodetection of the toxin in cytosol isolated from -injected rats and the use of cell-free endosomes. Thus, intact and -A were the only species detected in vivo in the soluble cytosolic fraction after toxin administration and in vitro in the extraendosomal medium during a cell-free translocation assay. However, we cannot exclude the possibility that a small number of fragments generated by endosomal cathepsins B and D physiologically translocate from the endosomal lumen to the cytoplasm and interact with cytosolic targets (EF-2 and mitochondria). Low production and or translocation of fragments, as well as short halflives in the cytosolic compartment, may well explain why they were not detected. Alternatively, the processed fragments may have lost structural elements essential for translocation across the endosomal membrane. On the other hand, endosomal proteolysis of may represent a degradative pathway related to the deactivation and termination of intracellular toxin cytotoxicity. Clearly, further studies are required to determine whether fragments generated by endosomal cathepsins B and D fully participate in the cytotoxic action of in hepatic tissue. Intravenously injected is taken up efficiently by the liver at an early time after death (5 min postinjection), suggesting a high binding capacity of in hepatic parenchyma. Indeed, injection of the toxin into mice has been shown to result in an early and profound inhibition of hepatic protein synthesis [27]. Our results suggest that a 2 MG LRP1 contributes, at least in part, to endocytosis in rat liver in vivo, based on the following: (a) the injection of a 2 MG, which partially reduced the endosomal association and processing of coinjected ; and (b) a time-dependent increase in immunodetectable a 2 MG LRP1 in hepatic endosomes induced by the toxin injection. It has been proposed that proteolytic nicking of at the Arg279-Glu280 peptide bond mediated by furin activity is at least partly required for expression of cytotoxicity [2,13]. In the present study, our observation that -A associates with hepatic plasma membrane, endosomal and cytosolic fractions isolated from -injected rats is consistent with this view. However, our in vivo and in vitro data also support the contention that the furin-mediated conversion of native into -A within hepatic endosomes may represent a minor metabolic fate for the internalized toxin, based on the following: (a) the lack of sensitivity of endosomal -degrading activity to furin inhibitors [hexa-d-arginine (HA)]; and (b) the predominant association of low molecular mass fragments of -A with hepatic endosomes at a late stage of endocytosis (60 min post- treatment). Finally, our data suggesting the presence of intact and -A at the cell surface are consistent with the endocytosis of native (major pathway) as well as -A (minor pathway) from the cell surface to early endosomes [28,29]. FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3743

10 Proteolysis of in rat hepatic endosomes T. El Hage et al. In the present work, we showed that the endosomal acidic proteolytic activity directed towards the internalized was comparable to that of the cysteine protease cathepsin B and the aspartic acid protease cathepsin D, as indicated by the following observations: (a) the inhibitor profile of the endosomal -degrading activity was very similar to that of cathepsins B and D [30]; and (b) the endosomal activity produced a substrate cleavage pattern that was very similar to that generated with pure cathepsins B or D. Interestingly, previous studies have shown that intracellular processing of by a PA-sensitive protease was critical for -induced lymphoproliferation, confirming that one or more intracellular proteases distinct from furin participate in processing within toxin-treated cells [31]. Moreover, additional metallo-dependent proteolytic activities (EDTA-sensitive) might act on internalized within endosomes and produce fragments with a molecular mass very close to that of intact. All cleavages produced by cathepsins B and D in the toxin are located within -A. A major degradation product of results from proteolytic cleavage at Thr396-Cys397 in the C-terminal extremity of domain I or Ib. The degradation product contains the entire catalytic -A domain (amino acids ) extended at the N-terminus by the CPV tripeptide, and may represent the main catalytic fragment responsible for the ADP-ribosyltransferase activity identified in vitro after cathepsin treatment. Three degradation products (peptides 4a, 4b and 4d) displayed a molecular mass slightly less than that of the native 66 kda and the unmodified N-terminal sequence, suggesting the removal of the C-terminal residues of encompassing the REDLK sequence. However, an antibody that recognizes the REDLK-mediated ER retrieval motif, which is located at the C-terminus of -A, showed immunoreactivity with endosomal -A, suggesting that the REDLK motif was not completely lost from -A within endosomes. It has previously been shown that human serum contains a carboxypeptidase activity, suggested to be carboxypeptidase-n, carboxypeptidase-h or carboxypeptidase-m, which removed the C-terminal Lys of and generated a processed form of ending in REDL [15]. We have now extended these observations to the endosomal apparatus, and suggest that may undergo C-terminal processing that begins early in the circulating blood, and is continued later within endosomes after entry of the toxin into the cell. Western blot analyses of associated with hepatic subcellular fractions under nonreducing conditions showed that the Cys265 Cys287 disulfide bridge was partially cleaved at the plasma membrane, endosome and cytosol loci. Thus, as for the proteolytic cleavage of at the connecting A B junction bond, the hepatic -reducing activity may well operate early at the cell surface prior to endocytosis. Moreover, the level of reduction within hepatic endosomes was much lower than that of proteolysis, suggesting that the endosomal reductive pathway may represent a minor metabolic fate for the internalized toxin [32]. It has been previously suggested that reduction is a two-step process: toxin unfolding that allows access to the Cys265 Cys287 bond is followed by reductive cleavage of the disulfide bond by a protein disulfide isomerase-like enzyme [14]. Importantly, toxin unfolding and reducing activities were present in the membrane fraction of toxin-sensitive cells but not in the soluble fraction, suggesting that the cytosol and the endosomal lumen may not be the relevant compartments for such cell-mediated reducing events [14]. One endosome-located mechanism that regulates activation and action occurs at the level of organelle acidification [33]. First, a low ph has been proposed to be required for the proteolytic cleavage of by furin [34]. Thus, whereas furin displays an optimal ph of 7 for model peptide substrates [35], the proteolysis of by furin is maximal between ph 5.0 and ph 5.5 [34]. Moreover, the vacuolar H + -ATPase inhibitor bafilomycin protected mouse L cells from the toxic effects of intact as well as precleaved, suggesting that an acidic environment is required for proteolytic activation of and additional event(s) leading to its cytotoxic effect [33]. Finally, it has clearly been shown that endosomal acidity facilitates the binding of to the endosomal membrane of mouse L cells (maximal binding observed at ph 4.0) and -induced pore formation in the lipid bilayer of endosomal vesicles (maximal effect at ph < 6) [8]. Our data showing the in vitro proteolysis of by endosomal acidic cathepsins and translocation of the internalized toxin across the endosomal membrane at low ph would be consistent with these prior observations. Other studies reported that translocation was strictly dependent on ATP hydrolysis but was not affected by bafilomycin, the H + -ATPase inhibitor [9]. These differences may result from the experimental approaches used (the rat liver in vivo model versus cellular in vitro systems) and or may be related to differences between hepatocytes and other cell types. In vivo [36] and in vitro [37] studies have shown that the normal airway epithelium is highly resistant to P. aeruginosa-induced apoptosis. Moreover, in airway target cells, induced a wide range of biochemical 3744 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

11 T. El Hage et al. Proteolysis of in rat hepatic endosomes and morphological changes (early mitochondrial dysfunction) that are not characteristic of apoptosis [23]. On the other hand, live P. aeruginosa bacteria can induce the apoptotic death of human conjunctiva epithelial Chang cells [38] and J774A.1 macrophages [39] through the type III secretion system. Also, -induced human mast cell apoptosis by activation of the caspase-8 and caspase-3 pathways and downregulation of antiapoptotic FLIP proteins has been reported [3]. In the present work, we have demonstrated that, after administration, rat hepatic cells undergo in vivo apoptosis through DNA fragmentation, mitochondrial release of cytochrome c, and activation of caspase-9 and caspase-3. By contrast, the receptor caspase-8-dependent pathway did not contribute to -induced apoptosis in rat liver cells in vivo. We speculate that, in the cytoplasm of toxin-treated hepatic cells, translocated can effect its cytotoxicity through a dual action, i.e. ADP-ribosylation of EF-2 (inhibition of protein synthesis) and mitochondrial alteration (intrinsic apoptotic effect). Both pathways require the translocation of into the cytoplasm of toxin-treated cells. On the basis of the reconstitution of the cytotoxic pathways with in vitro cytosol and cell-free mitochondria, our data suggest a direct interaction between and cytosolic EF-2 on the one hand, and the mitochondrial membrane on the other hand. However, the potential role (if any) of ADP-ribosylation of EF-2 in the mitochondrial apoptotic response induced by the toxin remains to be determined. Finally, we assign an important role to the endosomal acidic cathepsins B and D in increasing the in vitro transfer of the ADP-ribosyl moiety of NAD + to EF-2 by, but not in the release of cytochrome c from cell-free mitochondria. In summary, we found that internalized was rapidly proteolyzed within rat hepatic endosomes by cathepsins B and D, with subsequent ATP-dependent translocation of intact and -A to the cytosol. Intact induced ADP-ribosylation of cytosolic EF-2 as well as the mitochondrial release of cytochrome c, both in vivo and in vitro, with the in vitro effects being substantially increased by cathepsin B D pretreatment. Studies are currently underway to elucidate whether -induced mitochondrial alteration is mediated by the catalytic A-subunit or hydrophobic B-domain of, or whether it involves the dual heterogeneous part of the toxin. Use of this approach will provide novel insight(s) into the physiological significance of the endosomal fragments of internalized, which, until now, has remained elusive. Experimental procedures Peptides, enzymes, antibodies, protein determination, N-terminal sequencing, enzyme assays, and materials Pseudomonas aeruginosa, DT and bafilomycin-a1 were purchased from Calbiochem. Bovine cathepsin D (EC , 15 UÆmg )1 ), bovine cathepsin B (EC , 10 UÆmg )1 ), recombinant truncated human furin (2000 UÆmL )1 ) and human plasma a 2 MG were purchased from Sigma. Rabbit antibody against Pseudomonas was purchased from Sigma. Western blot analysis using the antibody against revealed a strong affinity for with a specificity for the A-subunit. Mouse monoclonal antibody directed against rat EEA1 was purchased from Transduction Laboratories. Mouse monoclonal antibody directed against rat cytochrome c was purchased from Pharmingen. Rabbit polyclonal antibody against human EF-2 and goat polyclonal antibody raised against the C-terminus of human LRP1 were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibody against KX 5 KDEL, which recognizes the ER retention signal KDEL and binds to various ER-resident proteins, was obtained from S. Fuller (EMBL, Heidelberg, Germany). Horseradish peroxidase-conjugated goat anti-(rabbit IgG) or goat anti-(mouse IgG) were from Sigma. The protein content of isolated fractions was determined by the method of Lowry et al. [40]. N-terminal sequence data were obtained by automated Edman degradation with a Procise sequencer (Applied Biosystems, Foster City, CA, USA), equipped with an on-line phenylthiohydantoin amino acid analysis system. Quantitative analysis of DNA fragmentation after toxin-induced cell death was analyzed by immunoassay determination of cytoplasmic histone-associated DNA fragments, according to the manufacturer s protocol (Roche). N-Acetyl-b-d-glucosaminidase was assayed with p-nitrophenyl N-acetyl-b-d-glucosaminide as substrate, according to Touster et al. [41]. Acid phosphatase was assayed as described by Trouet [42]. Caspase-3, caspase-8 and caspase-9 activity was analyzed with a fluorometric assay kit (BioVision, Mountain View, CA, USA) with the respective DEVD-AFC, IETD-AFC and LEHD-AFC substrates. Nitrocellulose membranes and the enhanced chemiluminescence detection kit were from Amersham. PA, E-64, phenylmethanesulfonyl fluoride and EDTA were from Sigma. HA was from Calbiochem. All other chemicals were obtained from commercial sources and were of reagent grade. Animals and injections In vivo procedures were approved by the institutional committee for use and care of experimental animals. Male Sprague-Dawley rats, body weight g, were obtained from Charles River France (St Aubin Les Elbeufs, FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 3745

12 Proteolysis of in rat hepatic endosomes T. El Hage et al. France) and fed ad libitum. Native or DT (15 lg per 100 g body weight) in 0.4 ml of 0.15 m NaCl was injected within 5 s into the penile vein under light anesthesia with ether. Rat liver subcellular fractionation Subcellular fractionation was performed using established procedures [22,24,25]. Following injection of toxins, rats were killed and livers were rapidly removed and minced in ice-cold isotonic homogenization buffer as previously described [22,24,25]. Rat liver large granule and cytosolic fractions were isolated by differential centrifugation as previously described [43 46]. Plasma membrane was prepared according to the method of Neville [47] as described by Authier et al. [43,48,49]. The endosomal fraction was isolated by discontinuous sucrose gradient centrifugation and collected at the m sucrose interface [22,24,25]. Endosomal fractions revealed no significant enrichment of lysosomal enzyme markers (N-acetyl-b-d-glucosaminidase, relative specific activity = 1.5; acid phosphatase, relative specific activity = 2.2), with the yield of enzymes accounting for < 0.2% of that of the homogenate. The recovery of organelle enzyme markers in the nonsedimentable cytosolic fraction was very low, and is in agreement with our previously published biochemical characterizations [43,50]. cathepsin-treated, according to Uren et al. [51]. In some experiments, KCl was omitted from the incubation medium to disrupt mitochondria by hypotonic lysis. Samples were incubated at 37 C for various periods (5 min to 2 h) and centrifuged for 15 min at g. Supernatants were subjected to reducing SDS PAGE followed by western blot analysis with antibody against cytochrome c. Immunoblot analysis Electrophoresed samples were transferred onto nitrocellulose membranes for 60 min at 380 ma in transfer buffer containing 25 mm Tris base and 192 mm glycine. The membranes were blocked by a 3 h incubation with 5% skimmed milk in 10 mm Tris HCl (ph 7.5), 300 mm NaCl and 0.05% Tween-20. The membranes were then incubated with primary antibody [mouse IgG against rat cytochrome c (diluted 1 : 1000), mouse monoclonal antibody against rat EEA1 (diluted 1 : 1000), rabbit polyclonal IgG against either (diluted 1 : ), KX 5 KDEL (diluted 1 : 100) [22] or human EF-2 (diluted 1 : 500)] in the above buffer for 16 h at 4 C. The blots were then washed three times with 0.5% skimmed milk in 10 mm Tris HCl (ph 7.5), 300 mm NaCl and 0.05% Tween-20 over a period of 1 h at room temperature. The bound immunoglobulin was detected with horseradish peroxidase-conjugated goat anti-(rabbit IgG) or goat anti-(mouse IgG). Cell-free proteolysis and translocation of endosome-associated Endosomal fractions isolated 30 min after the injection of native (15 lg per 100 g body weight) were suspended at 1 mgæml )1 in 0.15 m KCl, 5 mm MgCl 2 and 25 mm Hepes (ph 7) or 25 mm citrate phosphate buffer (ph 5 6) in the presence or absence of 10 mm ATP and 0.01 lm bafilomycin-a1. Samples were incubated at 37 C for various periods and subjected to reducing SDS PAGE followed by western blotting to determine the endosomal content and integrity of and -A. To specifically assess the membrane translocation of intact and processed through the endosomal membrane, incubation mixtures were centrifuged for 60 min at g. Pelleted endosomes and supernatants were then subjected to reducing SDS PAGE followed by western blot analysis with antibody against. Cell-free translocation of mitochondria-associated cytochrome c A rat liver mitochondrial fraction (large-granule fraction) was isolated by differential centrifugation as previously described [43,46], and then resuspended at 7.5 mgæml )1 in 0.15 m KCl, 5 mm MgCl 2, 1 mm EDTA and 10 mm Hepes (ph 7.5) in the presence or absence of native or In vitro proteolysis of by hepatic endosomes and proteases The endosomal fraction ( 1 lg) was incubated for varying lengths of time at 37 C with 10 lg of native in 30 ll of 25 mm citrate phosphate buffer (ph 5) or 25 mm Hepes buffer (ph 7) containing 6 mm CaCl 2, in the presence or absence of protease inhibitors. To determine the integrity of, the proteolytic reaction was stopped by the addition of reducing SDS PAGE sample buffer, and this was followed by SDS PAGE and western blot analysis. For some experiments, (10 lg) was digested in vitro for varying lengths of time with bovine cathepsin B or cathepsin D (5 UÆmL )1 Æmg )1 ) in 50 mm citrate phosphate buffer (ph 4 6), or human furin (10 UÆmL )1 Æmg )1 ) in 50 mm Hepes buffer (ph 7) containing 10 mm CaCl 2 and 10 mm dithiothreitol. The proteolytic reaction was stopped by the addition of reducing SDS PAGE buffer, and this was followed by SDS PAGE and Coomassie Brilliant Blue staining or western blot analysis. -catalyzed ADP-ribosylation of cytosolic EF-2 Native (10 lg) was incubated with 5 UÆmL )1 Æmg )1 cathepsins B and D at 37 C for 30 min in 25 mm citrate phosphate buffer (ph 4 6) and 0.1 m dithiothreitol in the presence or absence of 5 lgæml )1 PA and 1 lm E FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS