Abstract
BackgroundThe small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation.Methodology and Principal FindingsUsing cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking.Conclusions and SignificanceESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed.
Highlights
The plant toxin ricin and the bacterial Shiga and Shiga-like toxins (SLTx) bind their receptors at the mammalian cell surface and, after internalization by endocytosis, traffic in vesicular carriers to the endoplasmic reticulum (ER) [1] (Figure 1)
Eeyarestatin I (ESI) acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed
HeLa cells were pretreated with ESI or its inactive derivative ESR35 [38] for 1 h and challenged for 1, 2 or 4 h with dilutions of ricin or SLTx in medium containing ESI or ESR35 as appropriate, such that the compounds were present throughout the toxin challenge period
Summary
The plant toxin ricin and the bacterial Shiga and Shiga-like toxins (SLTx) bind their receptors (glycoproteins/glycolipids bearing exposed b1R4 linked galactose residues and the sphingolipid Gb3 respectively) at the mammalian cell surface and, after internalization by endocytosis, traffic in vesicular carriers to the endoplasmic reticulum (ER) [1] (Figure 1). Substrates are usually polyubiquitylated on internal lysine residues by membrane integral E3 ubiquitin ligases such as HRD1 This process provides tags for the cytosolic AAA (ATPase associated with various cellular activities)-ATPase extraction motor p97/ VCP [8], [9], [10] and subsequently, after limited de-ubiquitylation [11], [12], [13] for the proteasome [14], which is the terminal destination for ERAD substrates. The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. We investigated ESI as a potential inhibitor of toxin dislocation
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