An engineered TGF-\u3b2 monomer that functions as a dominant negative to block TGF-\u3b2 signaling

摘要

The transforming growth factor beta isoforms, TGF-\u3b21, -\u3b22, and -\u3b23 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-\u3b2 pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. In spite of the known importance of TGF-\u3b2s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-\u3b2 monomer, lacking the heel helix, a structural motif essential for binding the TGF-\u3b2 type I receptor, T\u3b2RI, but dispensible for binding the other receptor required for TGF-\u3b2 signaling, the TGF-\u3b2 type II receptor, T\u3b2RII, as an alternative therapeutic modality for blocking TGF-\u3b2 signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-\u3b2 monomers and bound T\u3b2RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-\u3b2 signaling with a Ki of 20 - 70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for T\u3b2RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T\u3b2RI, enabled it to bind endogenous T\u3b2RII, but prevented it from binding and recruiting T\u3b2RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-\u3b2 signaling, and may inform similar modifications of other TGF-\u3b2 family members.