title: Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC-MS.
abstract: The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20-30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.
authors.affiliation: Merck Serono S.A., Geneva Research Center, Department of Protein and Cell Sciences, 9 Chemin Des Mines, 1202 Geneva, Switzerland. vincent.dechavanne@merckgroup.com
authors.forename: Vincent
authors.initials: V
authors.lastname: Dechavanne
authors.forename: Francis
authors.initials: F
authors.lastname: Vilbois
authors.forename: Loic
authors.initials: L
authors.lastname: Glez
authors.forename: Bruno
authors.initials: B
authors.lastname: Antonsson
mesh_terms: D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000302:isolation & purification*; D021122:Protein Subunits; D014357:Trypsin / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D002918:Chymotrypsin; D014357:Trypsin; D020169:Caspases; D046988:Proteasome Endopeptidase Complex