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An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling, J. Biol. Chem., 292:7173-7188 (2017).
The TGF-βs, TGF-β1, -β2, and –β3, have essential roles regulating the adaptive immune system and maintaining the extracellular matrix. The dysregulation of the TGF-β pathway is however responsible for promoting the progression of several human diseases, including cancer and fibrosis. In spite of the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. To address the pressing need for novel TGF-b inhibitors with improved properties, we designed a TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor, TβRI, but dispensible for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor, TβRII, as an alternative therapeutic modality. 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.
Binding Properties of the Transforming Growth Factor-β Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling, Biochemistry, 55, 6880-6896 (2016).
TGF-β1, -β2, and -β3 each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor-receptor contact.
Biological activity differences between TGF-β1 and TGF-β3 correlate with differences in the rigidity and arrangement of their component monomers, Biochemistry, 53:5737-5749 (2014)
The three human TGF-β isoforms, TGF-β1, -β2, and β3, share 71-80% sequence identity and signal through the same receptors, yet the isoform-specific null mice have distinctive phenotypes and are inviable. The replacement of the coding sequence of TGF-β1 with TGF-β3 and TGF-β3 with TGF-β1 led to only partial rescue of the mutant phenotypes, suggesting that intrinsic differences between them contribute to the requirement of each in vivo. Here, we investigated whether the previously reported differences in the flexibility of the interfacial helix and arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues 54-75 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity. Conformational and activity differences were also observed between TGF-β3 and a variant with four helix-stabilizing residues from TGF-β1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as proposed.
Cooperative Assembly of TGF-β Superfamily Signaling Complexes is Mediated by Two Disparate Mechanisms and Distinct Modes of Receptor Binding, Mol. Cell 29:157-168 (2008)
See also A very private TGF-β receptor embrace, Mol. Cell 29:149-150 (2008)
Proteins of the TGF-b family signal across cell membranes in a distinctive manner by assembling heterotetrameric complexes of structurally-related serine/threonine-kinase receptor pairs. Unlike complexes of the bone morphogenetic protein (BMP) branch that assemble due to avidity from membrane localization, TGF-b complexes assemble cooperatively through recruitment of the low affinity (type I) receptor by the ligand-bound high affinity (type II) pair. In this study, the crystal structure of TGF-b3 in complex with the extracellular domains of both pairs of receptors was determined, revealing that the type I receptor docks and becomes tethered via novel extensions at a composite ligand-type II interface. Disrupting the receptor-receptor interactions conferred by these extensions abolishes assembly of the signaling complex and signal transduction (Smad activation). Although structurally similar, BMP and TGF-b receptors bind in dramatically different modes, mediating graded and switch-like assembly mechanisms that may have coevolved with branch-specific groups of cytoplasmic effectors.