title: Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6.
abstract: Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.
authors.affiliation: Discovery, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA. james.gavin@mpi.com
authors.forename: James M
authors.initials: JM
authors.lastname: Gavin
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Chen
authors.forename: Hua
authors.initials: H
authors.lastname: Liao
authors.forename: Neil
authors.initials: N
authors.lastname: Rollins
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Jingya
authors.initials: J
authors.lastname: Ma
authors.forename: Huay-Keng
authors.initials: HK
authors.lastname: Loke
authors.forename: Trupti
authors.initials: T
authors.lastname: Lingaraj
authors.forename: James E
authors.initials: JE
authors.lastname: Brownell
authors.forename: William D
authors.initials: WD
authors.lastname: Mallender
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Gould
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Amidon
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D011756:Diphosphates / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding / Q000187:drug effects; D018411:Spodoptera; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011756:Diphosphates; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; C521757:UBA6 protein, human; C109184:UBD protein, human; D014452:Ubiquitins; D000249:Adenosine Monophosphate; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D044764:Ubiquitin-Activating Enzymes