title: Absolute quantification of E1, ubiquitin-like proteins and Nedd8-MLN4924 adduct by mass spectrometry.
abstract: Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102-111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8-MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.
authors.affiliation: Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.forename: James E
authors.initials: JE
authors.lastname: Brownell
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Fengying
authors.initials: F
authors.lastname: Zhu
authors.forename: Jingya
authors.initials: J
authors.lastname: Ma
authors.forename: Huay-Keng
authors.initials: HK
authors.lastname: Loke
authors.forename: Neil
authors.initials: N
authors.lastname: Rollins
authors.forename: Teresa A
authors.initials: TA
authors.lastname: Soucy
authors.forename: James J
authors.initials: JJ
authors.lastname: Minissale
authors.forename: Michael P
authors.initials: MP
authors.lastname: Thomas
authors.forename: William D
authors.initials: WD
authors.lastname: Mallender
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Hua
authors.initials: H
authors.lastname: Liao
mesh_terms: D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000737:chemistry*; D005260:Female; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D000075687:NEDD8 Protein; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis; D011743:Pyrimidines / Q000737:chemistry*; D051381:Rats; D011923:Rats, Nude; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003517:Cyclopentanes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D010455:Peptides; D011743:Pyrimidines; D011994:Recombinant Proteins; C471538:UBA1 protein, human; D025801:Ubiquitin; D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human; C539933:pevonedistat