title: Covalent modification of the NF-κB essential modulator (NEMO) by a chemical compound can regulate its ubiquitin binding properties in vitro.
abstract: Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO's interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.
authors.affiliation: From the McArdle Laboratory for Cancer Research, Department of Oncology, Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, Wisconsin 53705 and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hooper
authors.affiliation: From the McArdle Laboratory for Cancer Research, Department of Oncology, Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, Wisconsin 53705 and Medical Scientist Training Program, and.
authors.forename: Shawn S
authors.initials: SS
authors.lastname: Jackson
authors.affiliation: Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Emma E
authors.initials: EE
authors.lastname: Coughlin
authors.affiliation: Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin 53706 Department of Chemistry, Department of Biomolecular Chemistry, and.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: From the McArdle Laboratory for Cancer Research, Department of Oncology, Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, Wisconsin 53705 and Medical Scientist Training Program, and smiyamot@wisc.edu.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology; D054358:Withanolides / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: C491731:IKBKG protein, human; D047908:Intracellular Signaling Peptides and Proteins; C514981:NEMO protein, mouse; D011994:Recombinant Proteins; D025801:Ubiquitin; D054358:Withanolides; D051550:I-kappa B Kinase; C009684:withaferin A
keywords: NF-kappa B (NF-κB); NF-κB Essential Modulator; Polyubiquitin Chain; Recombinant Protein Expression; Small Molecule; Withaferin A; Zinc Finger