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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME156AntigenAntigenProcessingProcessingandPresentationandPresentationProtocolsProtocolsEditedbyEditedbyJoyceC.SolheimJoyceC.SolheimHUMANAPRESSHUMANAPRESSProteasomePurification11Purificationof20SProtea
2、somesJillR.Beyette,TimothyHubbell,andJohnJ.Monaco1.IntroductionProteasomesarelargemulticatalyticproteinaseslocatedinthenucleiandcytoplasmofalleukaryoticcells.Proteasomesarecomposedoffourheptamericringsstackedtoformahollowcylinder(length16–20nm,diameter11–1
3、2nm).Theoutertworingsarecomposedofα-subunits,whileβ-subunits,whichcontaintheactivesites,comprisetheinnertworings.Proteasomesfromarchaebacteriacontainonlyonetypeeachofα-andβ-subunits.Eukaryoticproteasomesaremoredivergent;yeastproteasomeshavesevendifferentα-
4、andsevendifferentβ-subunits,eachoccupyingauniquepositioninthering.Onlythreeofthesevenyeastβ-subunitscontaintheN-terminalthreoninenec-essaryforactivity(1–2).Mammalian20Sproteasomeshavesevendifferentα-andtendifferentβ-subunits,andhavebeenclassifiedintotwogro
5、ups.Theso-called“constitu-tive”proteasomescontainthreecatalyticβ-subunits:PSMB5(XorMB1),PSMB6(Yorδ),andPSMB7(Z).Thesesubunitscanbereplacedin“immu-noproteasomes”bytheIFN-γ-induciblecatalyticβ-subunitsPSMB8(LMP7),PSMB9(LMP2),andPSMB10(MECL-1),respectively(1–
6、3).Althoughthereareeightpossiblecombinationsofcatalyticsubunitsintheβrings,proteasomeswithmixturesofconstitutiveandimmunesubunitsarenotfavored(4).Replace-mentofconstitutivecatalyticsubunitswiththeIFN-γ-induciblesubunitshasbeenshowntochangetheproteasomeacti
7、vitiesagainstfluorogenicpeptideandproteinsubstrates(2,5).Ithasbeenshownthatproteasomesarerespon-sibleforgenerationofcytosolicpeptides7–13aminoacidsinlength,whicharepresentedoncellsurfacesinassociationwithmajorhistocompatibilitycom-plexclassI(MHC-I)molecule
8、s(1,3).TheIFN-γ-induciblesubunitsarenotessentialforMHC-Iantigenpresentation,butitisthoughtthattheadditionalFrom:MethodsinMolecularBiology,vol.156:AntigenProcessingandPresentationProtocolsEditedby:J.C.Solheim©
