Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP-ribosylating and apoptotic effects.
To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell-free hepatic endosomes, and pure in vitro protease assays. ETA 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 ETA, in vivo association of the catalytic ETA-A subunit and low molecular mass ETA-A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal 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 activation, 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.
El Hage
Tatiana
T
INSERM, Châtenay-Malabry, France.
Lorin
Séverine
S
Decottignies
Paulette
P
Djavaheri-Mergny
Mojgan
M
Authier
François
F
D036002:ADP Ribose Transferases / Q000378:metabolism* / Q000493:pharmacokinetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000378:metabolism* / Q000493:pharmacokinetics; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D005098:Exotoxins / Q000378:metabolism* / Q000493:pharmacokinetics; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology*; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D037521:Virulence Factors / Q000378:metabolism* / Q000493:pharmacokinetics; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002401:Cathepsin B; D002402:Cathepsin D; D002403:Cathepsins; D005098:Exotoxins; D020652:Peptide Elongation Factor 2; D010446:Peptide Fragments; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C533916:Ctsb protein, rat; C533924:Ctsd protein, rat