Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin.

Abstract	The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor alpha. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.
Authors	Edda Fiebiger, Christian Hirsch, Jatin M Vyas, Eva Gordon, Hidde L Ploegh, Domenico Tortorella
Journal	Molecular biology of the cell (Mol Biol Cell) Vol. 15 Issue 4 Pg. 1635-46 (Apr 2004) ISSN: 1059-1524 [Print] United States
PMID	14767067 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References	1-(4-chlorophenyl)-3-(3-(4-chlorophenyl)-5,5-dimethyl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-1-hydroxyurea 1-(5,5-dimethyl-3-naphthalen-1-yl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-3-naphthalen-1-yl-1-hydroxyurea Hydrazones Luminescent Proteins Membrane Proteins Multienzyme Complexes RNA-Binding Proteins Recombinant Fusion Proteins US11 protein, herpesvirus Ubiquitin Viral Proteins Green Fluorescent Proteins Cysteine Endopeptidases Proteasome Endopeptidase Complex Hydroxyurea
Topics	Catalysis Cell Line Cell Membrane (metabolism) Cysteine Endopeptidases (metabolism) Cytosol (metabolism) Endoplasmic Reticulum (metabolism) Flow Cytometry Genes, Reporter Green Fluorescent Proteins Humans Hydrazones (pharmacology) Hydroxyurea (analogs & derivatives, pharmacology) Immunoblotting Luminescent Proteins (metabolism) Membrane Proteins (chemistry) Microscopy, Confocal Microscopy, Fluorescence Microscopy, Immunoelectron Models, Chemical Multienzyme Complexes (antagonists & inhibitors, metabolism) Precipitin Tests Proteasome Endopeptidase Complex Protein Folding RNA-Binding Proteins (metabolism) Recombinant Fusion Proteins (metabolism) Subcellular Fractions Time Factors Ubiquitin (metabolism) Viral Proteins (metabolism)

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