An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

  1. Andrew P. Hinck2
  1. From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260,
  2. the Departments of ‡Biochemistry and Structural Biology and
  3. §Cell Systems and Anatomy,
  4. **Center for Innovative Drug Discovery, University of Texas Health Science Center, San Antonio, Texas 78229-3900, and
  5. the ‖National Research Council, Human Health Therapeutics Portfolio, Montréal, Quebec H4P 2R2, Canada
  1. ↵2 To whom correspondence should be addressed: Dept. of Structural Biology, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, Rm. 1035, 3501 Fifth Ave., Pittsburgh, PA 15260. Tel.: 412-648-8533; Fax: 412-648-9008; E-mail: ahinck{at}pitt.edu.

  1. Edited by Norma Allewell

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 a Ki of 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.

Footnotes

  • ↵1 Supported by training grants provided by the Cancer Prevention Research Institute in Texas Grant RP1450105 and American Heart Association Grant 15PRE25550015.

  • This work was supported in part by National Institutes of Health Grants GM58670 and CA172886 (to A. H.) and Robert A. Welch Foundation Grant AQ1842 (to A. H.). Additional support was provided by University of Texas Health Science Center Cancer Therapy and Research Center Macromolecular Structure and Interactions Core supported by National Institutes of Health Grant P30 CA54174 from NCI, University of Texas Health Science Center San Antonio (UTHSCSA) Center for Macromolecular Interactions Core Facility supported by the UTHSCSA and UTHSCSA/University of Texas San Antonio Center for Innovative Drug Discovery and High Throughput Screening Facility supported by National Institutes of Health Grant NCATS UL1 TR001120 (to M. J. H). A. P. H. and T. S. are co-inventors of a provisional patent (United States Patent Application 62/423,920) that covers the dominant negative TGF-β, mmTGF-β2-7M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • This article was selected as one of our Editors' Picks.

  • This article contains Figs. S1–S8 and Table S1.

  • The atomic coordinates and structure factors (codes 5TX2, 5TX6, and 5TX4) have been deposited in the Protein Data Bank (http://wwpdb.org/).

  • The assigned chemical shifts for mmTGF-β2 and mmTGF-β2-7M were deposited to BioMagResBank (BMRB) under accession codes 26943 and 26944, respectively.

  • Received November 22, 2016.
  • Revision received February 12, 2017.
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  1. First Published on February 22, 2017 doi: 10.1074/jbc.M116.768754 The Journal of Biological Chemistry 292, 7173-7188.
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