Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA-A sub-unit and low molecular mass ETA-A fragments was observed in the endosomal apparatus.. We report
Trang 1endosomes by cathepsins B and D produces fragments
displaying in vitro ADP-ribosylating and apoptotic effects Tatiana El Hage1,2, Se´verine Lorin3, Paulette Decottignies4,5, Mojgan Djavaheri-Mergny6and
Franc¸ois Authier1,2
1 INSERM, Chaˆtenay-Malabry, France
2 Universite´ Paris-Sud, Faculte´ de Pharmacie, Chaˆtenay-Malabry, France
3 JE 2493, Universite´ Paris-Sud, Faculte´ de Pharmacie, Chaˆtenay-Malabry, France
4 CNRS, UMR 8619, Orsay, France
5 Universite´ Paris-Sud, Orsay, France
6 INSERM VINCO U916, Institut Bergonie´, Bordeaux, France
Keywords
cathepsin; endocytosis; endosome;
Pseudomonas exotoxin A; translocation
Correspondence
F Authier, INSERM, Universite´ Paris-Sud,
Faculte´ de Pharmacie, 5 rue Jean-Baptiste
Cle´ment, 92296 Chaˆtenay-Malabry, France
Fax: +33 1 46835844
Tel: +33 1 46835291
E-mail: francois.authier@u-psud.fr
(Received 21 March 2010, revised 4 June
2010, accepted 12 July 2010)
doi:10.1111/j.1742-4658.2010.07775.x
To assess Pseudomonas exotoxin A (ETA) compartmentalization, process-ing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administra-tion, cell-free hepatic endosomes, and pure in vitro protease assays ETA taken up into rat liver in vivo was rapidly associated with plasma mem-branes (5–30 min), internalized within endosomes (15–60 min), and later translocated into the cytosolic compartment (30–90 min) Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA-A sub-unit and low molecular mass ETA-A fragments was observed in the endosomal apparatus After an in vitro proteolytic assay with an endoso-mal lysate and pure proteases, the ETA-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 ETA-A Cell-free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA-A in vitro in a pH-dependent and ATP-dependent manner Rat hepatic cells underwent
in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activa-tion, and DNA fragmentation In an in vitro assay, intact ETA 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 ETA, involving cathepsins B and D, resulting in the production of ETA 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; ETA, exotoxin A;
HA, hexa- D -arginine; LRP1, low-density lipoprotein receptor-related protein 1; PA, pepstatin-A; SD, standard deviation;
a 2 MG, a2-macroglobulin.
Trang 2Exotoxin A (ETA) is considered to be the most toxic
factor secreted by Pseudomonas aeruginosa, a
Gram-negative opportunistic pathogen infecting
immunocom-promised individuals and burn victims [1] ETA is a
613 amino acid A⁄ B exotoxin that kills cells by
inhibi-tion of protein synthesis and programmed cell death
[2,3] ETA is secreted as a single polypeptide chain
composed of three structural and functional domains:
domain Ia (amino acids 1–252), which binds to the
a2-macroglobulin (a2MG)⁄ low-density lipoprotein
receptor-related protein 1 (LRP1) receptor present in
animal cells [4]; domain II (amino acids 253–364),
which contains a furin cleavage site
(Arg276-Gln277-Pro278-Arg279), the Cys265–Cys287 disulfide bond,
and a protein translocating sequence (amino
acids 280–313) [5,6]; and domain III (amino acids 400–
613), which contains the ADP-ribosylating enzyme [2]
To access and ADP-ribosylate its cellular target,
elongation factor-2 (EF-2), ETA must be transported
across the cellular membrane and into the cytoplasm
This is initiated by cell surface binding of ETA to
the a2MG⁄ LRP1 receptor [4], which is followed by
internalization of the toxin–receptor complex to the
endosomal apparatus by clathrin-dependent and
clath-rin-independent mechanisms [7] Two subcellular
com-partments have been proposed as being physiologically
relevant to the mechanism of translocation of
internal-ized ETA into the cytosol The first translocation
path-way has been proposed to operate at an early stage of
endocytosis from endocytic vesicles [8,9] Thus,
signifi-cant translocation of ETA across the endosomal
mem-brane of mouse lymphocytes was demonstrated, and
required exposure of ETA to low endosomal pH and
ATP hydrolysis [10] Other studies have proposed that
internalized ETA can be retrogradely transported to
the endoplasmic reticulum (ER) for retrotranslocation
to the cytosol through the Sec61 complex [11] The ER
trafficking pathway of ETA 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
ETA, activating processes have been proposed to occur
at various stages of ETA 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-ribosyla-tion of cytosolic EF-2, it was previously suggested that
intracellular production of a 37 kDa C-terminal ETA
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 proteo-lytic and reductive processing of ETA should be required for ETA cytotoxicity through the retrograde transport pathway [18], it has not been clearly deter-mined whether ETA requires proteolytic and⁄ or reduc-tive processing activation to reach the cytosol through the endosomal pathways and kill cells [19] Hence, recent studies have suggested that ETA 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 ETA 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 adminis-tration to rats and cell-free hepatic endosomes to relate the endosomal processing of internalized ETA to toxin cytotoxicity in a physiological state Following admin-istration of ETA to rats, rapid endocytosis of the intact unprocessed ETA was observed, coincident with the endosomal association of the ETA-A subunit (fast association) and low molecular mass ETA-A fragments (slow association) Our results assign an important role
to endosomal acidic cathepsins B and D in generating ETA fragments displaying high in vitro ADP-ribosyl-transferase activity towards cytosolic EF-2 We report
on the in vivo association of ETA and ETA-A with cytosolic fractions, and the in vitro ATP-dependent and pH-dependent translocation of ETA and ETA-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 frag-mentation were detected in cytosolic fractions isolated
2 h after ETA treatment, relating for the first time activation of the intrinsic apoptotic pathway with ETA cytotoxicity in a physiological state
Results
In vivo endocytosis and metabolic fate of ETA in rat liver
The kinetics of in vivo uptake of ETA at the hepatic cell surface (plasma membranes) (Fig 1A) and intra-cellularly (endosomes) (Fig 1B) were assessed first Rats were given an intravenous injection of native
Trang 3ETA (15 lg per 100 g body weight) and killed 5–90 min
postinjection Following preparation of hepatic
subcel-lular fractions, the amount of internalized ETA was
determined by SDS⁄ PAGE followed by western blot
analyses with antibody directed against ETA-A It was
assumed that the in vivo generation of free ETA-A was
attributable to both reductive and proteolytic cleavages
occurring within the ETA sequence Thus, both
processing pathways were analyzed, under either
non-reducing (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 ETA association with plasma
mem-branes was rapid (5 min postinjection) and maximal at
5–30 min postinjection, before decreasing with time
(Fig 1A) A transient association of ETA-A with
plasma membranes was also observed under reducing
and nonreducing conditions at 15–60 min postinjection
(Fig 1A) As compared with plasma membranes,
endosomal association of both ETA and ETA-A was
slightly delayed, with the maximum being observed at
15–60 min (ETA) or 30–90 min (ETA-A) (Fig 1B)
Low molecular mass ETA 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 ETA–
ETA receptor complex that is internalized into
toxin-treated cells, there are no published reports on the fate
of the ETA receptor during toxin endocytosis To
determine whether the ETA receptor was cointernal-ized along with the toxin, the in vivo effect of ETA treatment on the a2MG⁄ LRP1 receptor in the rat liver endosomal fraction was determined by immunoblotting (Fig 2A, upper blot) A high concentration of a mem-brane-bound 80 kDa fragment of LRP1 containing the tail epitope was found in the endosomal fraction iso-lated 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 ETA 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) How-ever, the level of the endosomal marker early endo-some antigen-1 (EEA1) was not modified after ETA treatment (Fig 2A, lower blot)
LRP1 enables endocytosis of ETA and various other ligands among such as a2MG [21] To examine the effect of a2MG on the uptake of ETA into hepatic endosomes, a2MG (15 lg per 100 g body weight) was coinjected with ETA into rats (Fig 2B) Endosomal association of intact ETA and ETA-A was reduced by
a2MG 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 chol-era toxin [22] As it was unknown whether antibodies
Plasma membranes
Nonreducing conditions
_ 5 15 30 60 90 (min, postinjection) _ 5 15 30 60 90
Endosomes
Nonreducing conditions
ETA
ETA ETA-A ETA-A
Reducing conditions
(66 kDa) ETA (66 kDa) ETA
Reducing conditions
– 100 – 75
– 100 – 75
(37 kDa) ETA-A (37 kDa) ETA-A
– 50 – 37 – 25
– 50 – 37 – 25
kDa – 15
kDa – 15
(min, postinjection)
Fig 1 Kinetics of appearance of ETA in hepatic plasma membranes and endosomes after toxin administration Rat hepatic plasma mem-brane (A) and endosomal fractions (B) were isolated at the indicated times after the in vivo administration of native ETA, 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 ETA 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 ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the right of the reducing blots The antibody against ETA also binds to undefined plasma membrane proteins dis-tinct from ETA 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 ETA-A.
Trang 4against KDEL bind to the ETA C-terminal sequence
REDLK (which resembles the ER motif KDEL), we
first characterized antibodies against KDEL for their
binding to native ETA and ETA-A by western blot
analysis (Fig 2C, left and middle blots) One antibody,
anti-KX5KDEL, bound to ETA-A but not to native
ETA (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 KX5KDEL was used
to assess the integrity of the REDLK peptide in
en-dosomal ETA-A under reducing conditions (Fig 2C,
right blot) KDEL immunoreactivity to internalized
ETA-A was detected in endosomal fractions isolated
from the livers of rats killed at 15–90 min postinjec-tion, with kinetics similar to those of ETA-A uptake into endocytic components (Fig 1B), suggesting that the C-terminal motif REDLK might not be completely removed from ETA-A within the endosomal apparatus
Endosomal proteolysis of internalized ETA by cathepsins B and D
To confirm the endosomal proteolysis of internalized ETA under conditions that maintained endosome integrity, we used cell-free endosomes containing
in vivo internalized ETA (Fig 3A,B) Endosomes were isolated 30 min following ETA injection, and intact endocytic vesicles were incubated for various times at
ETA _ 5 15 30 60 90 (min, postinjection)
α-LRP1 (tail)
LRP1 (80 kDa) 200
* *
100
_
5 15 30 60 90 (min, postinjection)
ETA ETA/ a 2 MG
15 30 5 15 30 (min, postinjection)
ETA-A
(37 kDa)
ETA
(66 kDa)
5
– 100 – 75
– 50 – 37
– 25
– 15
kDa
(180 kDa)
_
5 15 30 60 90 (min, postinjection)
ETA
5 15 30 60 ETA +
_
Furin
_
ETA
(66 kDa)
+
_
Dithiothreitol
ETA-A
(37 kDa)
ETA-A (37 kDa)
α-KX 5 KDEL α-KX 5 KDEL
(min, postinjection) 90
A
B
C
Fig 2 Characterization of ETA endocytosis into the endosomal apparatus (A) Changes in LRP1 concentration in the endosomal fraction following ETA injection into rats Hepatic endosomal frac-tions were isolated at the indicated times after the in vivo adminis-tration of native ETA, 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 ETA-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 ETA ⁄ 5 min or ETA ⁄ 15 min and control rats ( )) The arrows to the right indicate the mobilities of membrane-bound 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 a2MG treatment on the internalization of ETA Rat hepatic endosomal fractions were iso-lated at the indicated times after the in vivo coadministration of ETA and a2MG (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 ETA Each lane contained 50 lg of endosomal protein The arrows to the left indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the right (C) Assessment of immunoreac-tivity of antibody against KDEL for native and internalized ETA ETA was either untreated (left blot, lane )) or digested in vitro with
100 UÆmL)1Æmg)1 furin and 10 m M 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 ETA-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 ETA, and evaluated by western blotting for their immunoreactivity with polyclonal antibody against KX 5 KDEL (blot on the right) [22] The antibody against KX5KDEL also binds to undefined endosomal proteins distinct from ETA, 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 mobili-ties of intact ETA ( 66 kDa) and ETA-A ( 37 kDa) are indicated.
Trang 5neutral pH (pH 7) and 37C in an isotonic buffer
(which mimicked the intracellular milieu) in the
pres-ence or abspres-ence of ATP, the substrate of the vacuolar
H+-ATPase pump (Fig 3A) Immunoblot analyses
showed a progressive loss of intact ETA and ETA-A
in the presence of ATP, with concomitant generation
of ETA and ETA-A fragments Incubation in the
absence of ATP revealed a small amount of degrada-tion for intact ETA, whereas no degradadegrada-tion was observed for ETA-A (Fig 3A)
We next examined the effects of various protease inhibitors on the proteolysis of endosomal ETA and ETA-A, using cell-free endosomes preloaded with ETA toxin in vivo and incubated in vitro at pH 7 in the pres-ence of ATP (Fig 3B) Western blot analysis with the antibody against ETA revealed that the endosomal ETA-degrading activity was partially inhibited by the aspartic acid protease inhibitor pepstatin-A (PA), the cysteine protease inhibitor E64, and the metallopro-tease inhibitor EDTA
The inhibition of ETA-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 ETA by pure cathepsins B and D at pH 4–7 (Fig 3C)
pH 7 + ATP
_ PMSF PA EDTA HA E64
_
(Inhibitor)
(37 kDa) ETA-A
(66 kDa) ETA
ETA + Cath-D ETA + Cath-B
15 60 15 60 15 60 15 60 15 60 15 60 (min)
_
ETA (66 kDa) ETA-A (37 kDa)
ETA-A (37 kDa) ETA (66 kDa)
100 –
75 –
50 –
37 –
25 –
kDa
15 –
150
ETA ETA-A subunit
0 0 30 30 30 30 (min)
50
100
A
B
C
Fig 3 Assessment of ETA-degrading activity associated with hepatic endosomes (A) Rat hepatic endosomal fractions were iso-lated 30 min after ETA 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 m M Hepes (pH 7), 5 m M MgCl 2 , and 6 m M CaCl2, in the presence or absence of 10 m M ATP The integrity of ETA was then evaluated by reducing SDS ⁄ PAGE fol-lowed by western blotting with the polyclonal antibody against ETA Each lane contained 50 lg of endosomal protein The arrows
to the right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the left ETA and ETA-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 ETA administration (15 lg per
100 g body weight) and incubated at 37 C in isotonic buffer con-taining 0.15 M KCl, 25 m M Hepes (pH 7), 5 m M MgCl 2 , 6 m M CaCl 2 and 10 m M ATP for the indicated times in the presence or absence (lane )) of 2 m M phenylmethanesulfonyl fluoride (PMSF),
10 lgÆmL)1PA, 5 m M EDTA, 10 l M HA, or 10 l M E64 The integrity
of ETA was then evaluated by reducing SDS ⁄ PAGE followed by western blotting with the polyclonal antibody against ETA Each lane contained 50 lg of endosomal protein The arrows to the left and right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments (C) Native ETA (10 lg) was incubated at 37 C with cathepsin D (Cath-D) or cathepsin B (Cath-B) (5 UÆmL)1Æmg)1) in 50 m M citrate ⁄ phosphate buffer (pH 4–6) or 50 m M Hepes (pH 7) in the presence of 10 m M CaCl 2 and 10 m M dithiothreitol for the indicated times The integrity
of ETA was then evaluated by reducing SDS ⁄ PAGE followed
by western blotting with the polyclonal antibody against ETA The arrows to the left and right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments.
Trang 6Western blot analysis with an antibody against ETA
showed that cathepsins B and D degraded ETA in a
pH-dependent manner, with maximal degradation
being observed at pH 4 The ETA 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 ETA
sequence at various pH values (Fig 4A,B) The
prote-olysis of ETA 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
deter-mined by N-terminal sequence analysis (Fig 4B)
Edman degradation of intermediates 4a, 4b, 4d, 6a
and 6c revealed the N-terminal sequence of ETA
(AEEAFDL), suggesting that the cleavage sites are
located within the C-terminal region of the toxin
N-terminal sequence analysis of ETA 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 ETA
We next determined the presence of ETA in cytosolic fractions prepared from ETA-injected rats, using wes-tern blot analysis (Fig 5A) The intact 66 kDa ETA toxin was strongly detected within cytosolic fractions
at 0.5–4 h postinjection, and lower but detectable immunoreactivity for ETA-A was observed at 1–4 h under both reducing and nonreducing conditions The translocation of endosomal ETA into the extraendoso-mal milieu was then assessed with intact endosomes isolated 30 min after the injection of ETA and incu-bated for 0–2 h in isotonic medium at 37C in the presence or absence of ATP (Fig 5B) Western blot analysis of the ETA associated with sedimentable endosomes showed progressive decreases in immunore-active ETA and ETA-A at acidic pH (pH 5) or at
pH 7 in the presence of ATP Concomitantly, immu-noreactive ETA (high level) and ETA-A (low level) were progressively detected in the extraendosomal milieu, confirming the translocation of ETA toxin across the endosomal membrane at acidic luminal pH
No ATP-dependent translocation of ETA was observed
in the presence of bafilomycin, the H+-ATPase inhibitor
– 100 – 75 kDa
ETA (66 kDa)
ETA
1
– 50 – 37 – 25
– 15
4b
4c
6c 4a
6b 6a
6d 4d
0.25 3 0.25 3 0.25 Incubation time (h)
AEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDN ALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGN QLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRL HFPEGGSLAALTAHQACHLPLETFTRHRQPR279
1
ETA-B
280 GWEQLEQCGYPVQRLVALYLAARLSWNQVDQV IRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLT CP VAAGECA GPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYV FVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGD PALA YGYAQDQEPDARGRIRNGA LLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWP ETA-A
LAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK613
A
B
Fig 4 Structural characteristics of ETA fragments generated by cathepsin B and cathepsin D (A) Native ETA (10 lg) was incubated with bovine cathepsin B or cathepsin D (5 UÆmL)1Æmg)1), or a mixture of both, at 37 C for the indicated times in 50 m M citrate ⁄ phosphate buffer (pH 4–6) The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue Staining The major deg-radation products generated at pH 4 (peptides 4a–c) or pH 6 (peptides 6a–d) were subjected to N-terminal sequence analysis (B) Sequences
of ETA-A and ETA-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.
Trang 7Potential role of cathepsins B and D in the cytotoxic activity of ETA towards cytosolic targets
We first examined whether, under conditions where ETA was processed by cathepsins B and D, a corre-sponding change in the toxin cytotoxicity towards cytosolic EF-2 would be observed (Fig 6A) ETA 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 [32P]NAD A low level of ADP-ribosylation of EF-2 was evident after addition of untreated ETA to the cytosolic frac-tion After treatment of ETA with cathepsins B and
D, EF-2 labeling was increased, especially under acidic conditions (pH 6 > pH 5 > pH 4) However, cathep-sin treatment of ETA in the presence of protease inhibitors revealed [32P]NAD-ribose incorporation into cytosolic EF-2 comparable to that observed in the absence of protease treatment
A role for mitochondria in ETA-induced cell death has been previously shown with the use of human air-way epithelial target cells [23] Consequently, we exam-ined cytochrome c release from cell-free mitochondria isolated from control rats and then treated with ETA
in vitro (Fig 6B, upper blots) Cytochrome c associa-tion with intact rat liver mitochondria persisted during the incubation in isotonic medium, despite small but detectable release at 15 min However, there was sub-stantial release of cytochrome c into the resulting mito-chondrial supernatant after the addition of native ETA
or ETA that had been pretreated with a mixture of cathepsins B and D No detectable release of cyto-chrome c was observed following treatment of mito-chondria 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 frac-tions isolated after the in vivo injection of ETA into rats were analyzed for their cytochrome c content by immu-noblot analysis (Fig 6B, lower blots) Low but detect-able immunoreactivity towards cytochrome c was observed in cytosol isolated from noninjected rats In response to ETA, 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 ETA-triggered pro-grammed cell death was then analyzed by incubating hepatic cytosol isolated from ETA-treated rats with fluorescent substrates specific for caspase-9, caspase-3 and caspase-8 (Fig 6C, open bars) Caspase-9 and
_ 0.5 1 2
Pellet (endosomal medium)
_ 0.5 1 2 (incubation, h)
Supernatant (extraendosomal medium)
Cytosol
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7
_ 0.5 1 1.5 2 4 (h, postinjection)
ETA
Nonreducing conditions
ETA-A
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7 + ATP
ETA-A
(37 kDa)
ETA
(66 kDa)
Reducing conditions
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 5
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7 + ATP + Bafilomycin
A
B
Fig 5 In vivo and in vitro assessment of the cytosolic
transloca-tion of endosomal ETA (A) Rat hepatic cytosolic fractransloca-tions were
iso-lated at the indicated times after the in vivo administration of native
ETA, 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 ETA Each lane
con-tained 30 lg of cytosolic protein The arrows to the left indicate the
mobilities of intact ETA ( 66 kDa) and ETA-A ( 37 kDa) (B)
Membrane translocation of toxin peptides in cell-free rat hepatic
endosomes containing in vivo internalized ETA The endosomal
fraction was isolated 30 min after the administration of ETA, and
then resuspended in 0.15 M KCl containing 5 m M MgCl 2 and, when
indicated, 50 m M Hepes (pH 7), 50 m M citrate ⁄ phosphate buffer
(pH 5), 10 m M ATP, and 1 l M bafilomycin After the indicated times
of incubation at 37 C, endosomes were sedimented by
ultracen-trifugation, and the pellets (endosome-associated material) and
supernatants (extraendosomal material) were evaluated for their
content of ETA peptides by reducing SDS ⁄ PAGE followed by
wes-tern blotting with the polyclonal antibody against ETA Equivalent
volumes of each subfraction (40 lL) were loaded onto each lane.
The arrows to the left indicate the mobilities of intact ETA
( 66 kDa) and ETA-A ( 37 kDa).
Trang 8no toxin
Native ETA Cathepsin-treated ETA
EF-2
(105 kDa)
Cathepsin-treated ETA + cytosol
4+i (pH of proteolysis)
ADP-ribosylation of EF-2
Cytochrome c
(15 kDa)
Cytochrome c
(15 kDa)
_ 5 15 _ 5 15 _ 5 15 (min of incubation)
Intact mitochondria Disrupted mitochondria
EF-2
100
200
_ 0.5 1 1.5 2 4 (h, postinjection)
Cytochrome c
(15 kDa)
ETA Caspase-9
Toxin
_ 7 6 5 4 4+i (pH of proteolysis)
(15 kDa)
1
Caspase-3
4
2
4
Caspase-8
2 1
_ 5 15 30 60 90 120 240(min, postinjection)
_
0.5 1 1.5 2 4 (h, postinjection)
0.5
A
C
D B
Fig 6 Assessment of cytotoxic activity of cathepsin-treated ETA towards cytosolic target and mitochondria (A) Native ETA (10 lg) was digested in vitro for 30 min at 37 C with a mixture of cathepsins B and D (5 UÆmL)1Æmg)1) in 25 m M Hepes (pH 7) or 25 m M citrate ⁄ phos-phate buffer (pH 4–6) containing 0.1 M dithiothreitol (DT) and, when indicated, 5 lgÆmL)1PA and 1 l M E64 (lane 4 + i) The treated ETA (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 l M [ 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 densi-tometry, and the ratio of [32P]EF-2 signal ⁄ nonradiolabeled EF-2 signal was expressed as a percentage (mean ± SD) of that of untreated ETA (lane ), 100%) (lower panel) *P < 0.05 for the differences between pH 5 or pH 4 and untreated cytosol (B) Upper blots: rat liver mitochon-dria (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 ETA 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 ETA 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 ETA-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 ETA-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.
Trang 9caspase-3 activity increased in rat liver cytosol 1.5–2 h
after the injection of ETA, 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
frac-tions 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 ETA administration into rats
paral-leled caspase-9 and caspase-3 activation, with DNA
fragmentation being observed 2 h after ETA injection
and remaining elevated up to 4 h (Fig 6D, open bars)
No DNA fragmentation was observed in hepatic
cyto-solic fractions isolated from DT-injected rats (Fig 6D,
closed bars)
Discussion
Using the in situ liver model system, we have
previ-ously shown that, after cholera toxin binding to
hepa-tic cells, cholera toxin accumulates in a low-density
endosomal compartment and then undergoes
endoso-mal proteolysis by the aspartic acid protease
cathep-sin D [22,25] Ucathep-sing a similar methodology, others
have previously shown that the plant toxin ricin
fol-lows 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 ETA, another
A⁄ B toxin functionally related to DT that has an
iden-tical intracellular target (cytosolic EF-2) [6] Our data
clearly show that internalized ETA is susceptible to
hydrolysis by cathepsins B and D, which are present in
hepatic endosomes and operate at acidic pH
Compa-rable to the endosomal degradation of internalized CT
[22,25] and ricin [26] in rat hepatic endosomes, the
endosomal processing of internalized ETA occurred
mainly (if not totally) following ATP-dependent
acidi-fication of the endosomal lumen
Cytosolic translocation of endosomal ETA was
established through the immunodetection of the toxin
in cytosol isolated from ETA-injected rats and the use
of cell-free endosomes Thus, intact ETA and ETA-A
were the only ETA 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 ETA 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 ETA fragments, as well as short half-lives in the cytosolic compartment, may well explain why they were not detected Alternatively, the pro-cessed fragments may have lost structural elements essential for translocation across the endosomal mem-brane On the other hand, endosomal proteolysis of ETA may represent a degradative pathway related to the deactivation and termination of intracellular toxin cytotoxicity Clearly, further studies are required to determine whether ETA fragments generated by en-dosomal cathepsins B and D fully participate in the cytotoxic action of ETA in hepatic tissue
Intravenously injected ETA is taken up efficiently by the liver at an early time after death (5 min postinjec-tion), suggesting a high binding capacity of ETA in hepatic parenchyma Indeed, injection of the toxin into mice has been shown to result in an early and pro-found inhibition of hepatic protein synthesis [27] Our results suggest that a2MG⁄ LRP1 contributes, at least
in part, to ETA endocytosis in rat liver in vivo, based
on the following: (a) the injection of a2MG, which par-tially reduced the endosomal association and process-ing of coinjected ETA; and (b) a time-dependent increase in immunodetectable a2MG⁄ LRP1 in hepatic endosomes induced by the toxin injection
It has been proposed that proteolytic nicking of ETA at the Arg279-Glu280 peptide bond mediated by furin activity is at least partly required for expression
of ETA cytotoxicity [2,13] In the present study, our observation that ETA-A associates with hepatic plasma membrane, endosomal and cytosolic fractions isolated from ETA-injected rats is consistent with this view However, our in vivo and in vitro data also sup-port the contention that the furin-mediated conversion
of native ETA into ETA-A within hepatic endosomes may represent a minor metabolic fate for the internal-ized toxin, based on the following: (a) the lack of sensitivity of endosomal ETA-degrading activity to furin inhibitors [hexa-d-arginine (HA)]; and (b) the predominant association of low molecular mass frag-ments of ETA-A with hepatic endosomes at a late stage of ETA endocytosis (60 min post-ETA treat-ment) Finally, our data suggesting the presence of intact ETA and ETA-A at the cell surface are consis-tent with the endocytosis of native ETA (major pathway) as well as ETA-A (minor pathway) from the cell surface to early endosomes [28,29]
Trang 10In the present work, we showed that the endosomal
acidic proteolytic activity directed towards the
internal-ized ETA was comparable to that of the cysteine
pro-tease cathepsin B and the aspartic acid propro-tease
cathepsin D, as indicated by the following
observa-tions: (a) the inhibitor profile of the endosomal
ETA-degrading activity was very similar to that of
cathepsins B and D [30]; and (b) the endosomal
activ-ity produced a substrate cleavage pattern that was very
similar to that generated with pure cathepsins B or D
Interestingly, previous studies have shown that
intra-cellular processing of ETA by a PA-sensitive protease
was critical for ETA-induced lymphoproliferation,
confirming that one or more intracellular proteases
distinct from furin participate in ETA processing
within toxin-treated cells [31] Moreover, additional
metallo-dependent proteolytic activities
(EDTA-sensi-tive) might act on internalized ETA within endosomes
and produce fragments with a molecular mass very
close to that of intact ETA
All cleavages produced by cathepsins B and D in the
ETA toxin are located within ETA-A A major
degra-dation product of ETA results from proteolytic
cleav-age at Thr396-Cys397 in the C-terminal extremity of
domain I or Ib The degradation product contains the
entire catalytic ETA-A domain (amino acids 400–613)
extended at the N-terminus by the CPV tripeptide, and
may represent the main catalytic fragment
respon-sible for the ADP-ribosyltransferase activity identified
in vitro after cathepsin treatment Three degradation
products (peptides 4a, 4b and 4d) displayed a
molecu-lar mass slightly less than that of the native 66 kDa
ETA and the unmodified N-terminal ETA sequence,
suggesting the removal of the C-terminal residues of
ETA encompassing the REDLK sequence However,
an antibody that recognizes the REDLK-mediated ER
retrieval motif, which is located at the C-terminus of
ETA-A, showed immunoreactivity with endosomal
ETA-A, suggesting that the REDLK motif was not
completely lost from ETA-A within endosomes It has
previously been shown that human serum contains a
carboxypeptidase activity, suggested to be
carboxypep-tidase-N, carboxypeptidase-H or carboxypeptidase-M,
which removed the C-terminal Lys of ETA and
gener-ated a processed form of ETA ending in REDL [15]
We have now extended these observations to the
endosomal apparatus, and suggest that ETA may
undergo C-terminal processing that begins early in the
circulating blood, and is continued later within
endo-somes after entry of the toxin into the cell
Western blot analyses of ETA associated with
hepa-tic 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 ETA at the connecting A⁄ B junction bond, the hepatic ETA-reducing activity may well operate early
at the cell surface prior to ETA endocytosis More-over, the level of ETA reduction within hepatic endo-somes 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 ETA 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 pro-tein 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 com-partments for such cell-mediated reducing events [14] One endosome-located mechanism that regulates ETA activation and action occurs at the level of orga-nelle acidification [33] First, a low pH has been pro-posed to be required for the proteolytic cleavage of ETA by furin [34] Thus, whereas furin displays an optimal pH of 7 for model peptide substrates [35], the proteolysis of ETA 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 ETA as well as precleaved ETA, suggesting that an acidic environment
is required for proteolytic activation of ETA and addi-tional event(s) leading to its cytotoxic effect [33] Finally, it has clearly been shown that endosomal acid-ity facilitates the binding of ETA to the endosomal membrane of mouse L cells (maximal binding observed
at pH 4.0) and ETA-induced pore formation in the lipid bilayer of endosomal vesicles (maximal effect at
pH < 6) [8] Our data showing the in vitro proteolysis
of ETA by endosomal acidic cathepsins and transloca-tion of the internalized toxin across the endosomal membrane at low pH would be consistent with these prior observations Other studies reported that ETA 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, ETA induced a wide range of biochemical