Cell Microbiol. 2016 Dec;18(12):1800-1814. doi: 10.1111/cmi.12621.

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B-chain.

Authier F1, Djavaheri-Mergny M2, Lorin S3, Frénoy JP4, Desbuquois B5.

1 Service information scientifique et technique (IST) de l’Inserm, Paris, France.
2 Inserm U1218, Université de Bordeaux, Institut Bergonié, Bordeaux, France.
3 Inserm UMR-S-1193, Université Paris-Saclay, 92296, Châtenay-Malabry, France.
4 CNRS UMR 8601, Centre Universitaire des Saints-Pères, Université Paris-Descartes, Paris, France.
5 Inserm U 1016 and CNRS UMR 8104, Université Paris-Descartes, Institut Cochin, Paris, France.



Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.



Bacteria and plants produce a large variety of protein toxins which consist of two moieties: a B moiety that binds to cell surface components and allows toxin entry into the cell, and an enzymatically active A moiety that, after translocation from an intracellular membrane compartment, acts on a cytosolic target (Sandvig and Van Deurs, 1996, 2002; Sandvig et al, 2002, 2013; Spooner and Lord, 2012, 2015). Some toxins, such as anthrax and diphtheria toxins, enter the cytosol from endosomes, a process triggered by low pH-induced conformational change of the B subunit in the endosomal lumen and channel formation through the endosomal membrane. Other toxins, such as cholera and Shiga toxins, Pseudomonas exotoxin A and the plant toxin ricin, undergo retrograde transport from endosomes to the endoplasmic reticulum (ER) through the Golgi apparatus and translocation across the ER membrane, exploiting ER translocators normally involved in ER-associated protein degradation. Pharmacological (Stechman et al, 2010) and genetic (Moreau et al, 2011; Bassik et al, 2013) approaches have shown that retrograde translocation of these toxins to the ER is required for cytotoxicity. Apart cholera toxin, which activates adenylate cyclase via ADP ribosylation of the heterotrimeric Gs protein, most bacterial and plant toxins inhibit protein synthesis. Some toxins, such as Shiga toxin and ricin, inactivate ribosomes by removing a single adenine residue from the 28S RNA of the ribosomal 60S subunit, whereas others, such as diphtheria toxin and Pseudomonas exotoxin A, activate ADP-ribosylation of elongation factor 2. In addition, Shiga toxin and ricin induce nuclear DNA damage (Brigotti et al, 2002), cell stress responses and programmed cell death via both intrinsic and extrinsic pathways of apoptosis (Tesh, 2012). Although a threat to human health, bacterial and plant toxins have been valuable tools to characterize cellular processes such as endocytosis and intracellular transport, and as a constituent of immunotoxins they have received medical applications (Sandvig et al, 2010; Alewine et al, 2015).

It has previously been shown that, in intact rat liver, access of cholera toxin (Merlen et al, 2005; El Hage et al, 2007) and Pseudomonas exotoxin A (El Hage et al, 2010) to their cytosolic targets occurs from endosomes, in which these toxins are processed to biologically active fragments by acidic proteases. In the present study, we have examined whether this intracellular pathway also operates for ricin, which had been shown to undergo endocytosis in liver (Frénoy et al, 1992; Brech et al, 1998) and liver cells (Magnusson and Berg, 1993), and translocation across the endosomal membrane in cell-free endosomes of lymphocytes (Beaumelle et al, 1993). Using cell fractionation, SDS PAGE and Western immunoblotting procedures, we show that, following injection to rats, ricin taken up into the liver rapidly accumulates in endosomes as an intact A/B heterodimer and is later partially translocated to cytosol as A- and B-chains. Endocytosis of ricin is accompanied by the co-internalization of multiple membrane-associated ricin binding proteins. Unlike cholera toxin A- and B-subunits and diphtheria toxins, neither in intact cell-free endosomes containing in vivo internalized ricin nor in endosomal lysates incubated with ricin are ricin A- and B-chains processed to low molecular weight fragments in vitro. In cell-free endosomes containing in vivo internalized ricin, endosome-associated heterodimeric ricin and to a lesser extent ricin A- and B-chains are time-dependently released in the extra-endosomal medium, indicating a translocation of luminal ricin across the endosomal membrane. Endosome-localized thioredoxin reductase-1 (TrxR1) is required for ricin translocation as shown by its association with ricin chains upon co-immunoprecipitation of endosomal lysates. In addition, ricin translocation does not occur in endosomes depleted of TrxR1 or in the presence of cis-retinoic acid, an inhibitor of TrxR1 activity. Taken together, these observations identify liver endosomes as a subcellular site from which, following endocytosis, ricin undergoes translocation into cytosol in vivo and across the endosomal membrane in a cell-free system. This process differs from ricin translocation from the ER, which requires ricin reduction (Spooner et al, 2004) and involves solely the A-chain. TrxR1 appears to be required for ricin translocation from endosomes, but whether it is also involved in ricin reduction by protein disulfide isomerase or thioredoxin (Spooner et al, 2004; Bellisolla et al, 2004) at the cytosolic side of the endosomal membrane remains to be established.

Ricin has been shown to induce apoptosis in a variety of cells and organs, including liver, by activating both the intrinsic (mitochondria-mediated) and extrinsic (death receptor-mediated) pathways of apoptosis, but mainly the former (Tesh, 2012). An apoptotic effect of ricin B-chain in cultured cells has also been reported (Hasegawa et al, 2000). We show here that, following injection to rats, ricin induces a time- and dose-dependent increase in DNA fragmentation, caspase-9 and caspase-3 activity in liver, and a release of cytochrome c into cytosol, indicating activation of the mitochondria-mediated pathway of apoptosis. In addition, reduced ricin and ricin B-chain also induce a release of cytochrome c into liver cytosol, suggesting that ricin B-chain, rather than A-chain, mediates the induction of apoptosis by heterodimeric ricin. Furthermore, addition of reduced ricin and ricin B-chain to intact cell-free mitochondria induces the release of cytochrome c in the extra-mitochondrial medium, whereas under similar conditions no such effect is observed with diphtheria and cholera toxins. Taken together, these findings suggest that, following translocation of endocytosed ricin into cytosol, a direct interaction of ricin B-chain with some component of the outer mitochondrial membrane mediates the apoptotic effect of heterodimeric ricin. Interestingly, the B subunit of Shiga toxin Stx1 has been shown to also induce apoptosis in cultured cells (Mangeney et al, 1993; Nakagawa et al, 1999), as does the B chain of the plant toxin abrin (Ohba et al, 2004).



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