title: Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Using cytotoxicity measurements, we found no role for ES(I) 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
  ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Mina-Olga
authors.initials: MO
authors.lastname: Aletrari
authors.forename: Craig
authors.initials: C
authors.lastname: McKibbin
authors.forename: Helen
authors.initials: H
authors.lastname: Williams
authors.forename: Vidya
authors.initials: V
authors.lastname: Pawar
authors.forename: Paola
authors.initials: P
authors.lastname: Pietroni
authors.forename: J Michael
authors.initials: JM
authors.lastname: Lord
authors.forename: Sabine L
authors.initials: SL
authors.lastname: Flitsch
authors.forename: Roger
authors.initials: R
authors.lastname: Whitehead
authors.forename: Eileithyia
authors.initials: E
authors.lastname: Swanton
authors.forename: Stephen
authors.initials: S
authors.lastname: High
authors.forename: Robert A
authors.initials: RA
authors.lastname: Spooner
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism / Q000633:toxicity; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D006918:Hydroxyurea / Q000031:analogs & derivatives* / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D012276:Ricin / Q000378:metabolism / Q000633:toxicity; D022621:Shiga Toxin / Q000378:metabolism / Q000633:toxicity; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000378:metabolism
chemical_list: C492847:1-(4-chlorophenyl)-3-(3-(4-chlorophenyl)-5,5-dimethyl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-1-hydroxyurea; D004167:Diphtheria Toxin; C028391:G protein, vesicular stomatitis virus; D006835:Hydrazones; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D022621:Shiga Toxin; D012276:Ricin; D006918:Hydroxyurea