title: An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling.
abstract: The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with aKiof 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Sun Kyung
authors.initials: SK
authors.lastname: Kim
authors.affiliation: Cell Systems and Anatomy.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Barron
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Hinck
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Elyse M
authors.initials: EM
authors.lastname: Petrunak
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Cano
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Avinash
authors.initials: A
authors.lastname: Thangirala
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Brian
authors.initials: B
authors.lastname: Iskra
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Molly
authors.initials: M
authors.lastname: Brothers
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Machell
authors.initials: M
authors.lastname: Vonberg
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Belinda
authors.initials: B
authors.lastname: Leal
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Blair
authors.initials: B
authors.lastname: Richter
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Ravindra
authors.initials: R
authors.lastname: Kodali
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Taylor
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Shoucheng
authors.initials: S
authors.lastname: Du
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Barnes
authors.affiliation: the National Research Council, Human Health Therapeutics Portfolio, Montréal, Quebec H4P 2R2, Canada.
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: P John
authors.initials: PJ
authors.lastname: Hart
authors.affiliation: Center for Innovative Drug Discovery, University of Texas Health Science Center, San Antonio, Texas 78229-3900, and.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hart
authors.affiliation: the Departments of Biochemistry and Structural Biology and.
authors.forename: Borries
authors.initials: B
authors.lastname: Demeler
authors.affiliation: From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, ahinck@pitt.edu.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Hinck
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D018450:Disease Progression; D005109:Extracellular Matrix / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D020033:Protein Isoforms; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D020349:Surface Plasmon Resonance; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D020033:Protein Isoforms; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I
keywords: cancer; cell signaling; dominant negative; fibrosis; inhibitor; protein engineering; transforming growth factor beta (TGF-B)