# Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC
## 3 Discussion  
The most striking result in the present article was the ability of the compound EACC to induce a strong protection against ricin, as well as against other plant toxins. The data indicate that for the toxin we focused on in this study, ricin, this is mainly due to an inhibition of release of ricin A-chain in the ER, possibly also coupled to a decrease in translocation from the ER to the cytosol, since the protection seems to be stronger than the inhibition of reductive release of A-chain from the B-chain. Due to the sensitivity of the assay, we could not measure release after short times of incubation, raising the question of whether there is a change in the kinetics of reduction and translocation of the A-chain that might explain the larger effect on toxicity than on A-chain release measured after 3 h. EACC had little effect on endocytic uptake of ricin, there did not seem to be any strong effect on transport to the Golgi, and the results indicated that transport between the trans-Golgi and the ER was unchanged. Clearly, the data showed that a sulfated ricin construct, which also contained an ER glycosylation site, was modified by ER enzymes. As expected, there was no further modification of a ricin construct without this glycosylation site. In agreement with our data showing that EACC had very little effect on ricin endocytosis and degradation in HEp-2 cells is the reported lack of effect of EACC on EGF-induced degradation of the EGF-receptor and the lack of conversion of procathepsin B to mature cathepsin B in HeLa cells [7]. Since EACC was reported to inhibit the association of autophagosomes with Stx17 [7] and this SNARE has to be phosphorylated by TBK1 to initiate autophagy [8], we tested whether inhibition of TBK1 with MRT68601 would affect the protection afforded by EACC. As shown, it did counteract the protection, but this may not be related to the effect of EACC as the compound in itself sensitized the cells to ricin. Surprisingly, this sensitizing effect seemed to be due to a strong increase in transport of ricin from endosomes to the Golgi apparatus, whereas there was no further effect on transport from trans-Golgi to the ER. Importantly, addition of EACC gave a strong protection when added together with MRT68601 (compared to after addition of MRT68601 only), supporting the idea that EACC protects at a later step than transport to the ER. Thus, this is in agreement with the view that reduction and translocation of ricin is the step that is affected by EACC. The reason for the MRT68601-induced increased transport of ricin from endosomes to the Golgi is not obvious. However, it is now known that there are a number of common effectors in the endocytic and autophagic machinery [15], and it was found that TBK1 is acting upstream of recruitment of proteins involved in autophagy to endosomes [11]. Inhibition of recruitment of these factors was found to inhibit recycling of the EGF-receptor through the Rab11-positive recycling compartment, whereas no effect was found on the rate of uptake of the EGF-receptor or on uptake and recycling of the transferrin receptor. An effect of the TBK1-inhibitor MRT68601 on endosomal recruitment of proteins might be related to the change in transport of ricin to the Golgi apparatus. It is possible that when recycling is inhibited by inhibition of TBK-1, transport in the direction of the Golgi apparatus might be facilitated. In future studies, it would be valuable to investigate the endosomal protein complexes known to affect Golgi transport under these conditions and to study whether transport of other ligands and membrane components moving in the direction of the Golgi apparatus are also affected. An important question is why there is an inhibition of ricin A-chain release and translocation from the ER in the presence of EACC.