Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin.
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.
Fiebiger
Edda
E
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Hirsch
Christian
C
Vyas
Jatin M
JM
Gordon
Eva
E
Ploegh
Hidde L
HL
Tortorella
Domenico
D
D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D006918:Hydroxyurea / Q000031:analogs & derivatives* / Q000494:pharmacology; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
C492847:1-(4-chlorophenyl)-3-(3-(4-chlorophenyl)-5,5-dimethyl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-1-hydroxyurea; C492848:1-(5,5-dimethyl-3-naphthalen-1-yl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-3-naphthalen-1-yl-1-hydroxyurea; D006835:Hydrazones; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C074546:US11 protein, herpesvirus; D025801:Ubiquitin; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D006918:Hydroxyurea