Publications by authors named "Andrew P Hinck"

68 Publications

Distinct intramolecular interactions regulate autoinhibition of vinculin binding in αT-catenin and αE-catenin.

J Biol Chem 2021 Mar 23:100582. Epub 2021 Mar 23.

Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh PA 15261. Electronic address:

α-Catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate αE(epithelial)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of αT(testes)-catenin, an α-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that αT-catenin binds the β-catenin/N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.
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http://dx.doi.org/10.1016/j.jbc.2021.100582DOI Listing
March 2021

Structure and Role of BCOR PUFD in Noncanonical PRC1 Assembly and Disease.

Biochemistry 2020 07 10;59(29):2718-2728. Epub 2020 Jul 10.

Department of Biochemistry and Molecular Genetics, Midwestern University, 19555 North 59th Avenue, Glendale, Arizona 85308, United States.

Polycomb repression complex 1 (PRC1) is a multiprotein assembly that regulates transcription. The Polycomb group ring finger 1 protein (PCGF1) is central in the assembly of the noncanonical PRC1 variant called PRC1.1 through its direct interaction with BCOR (BCL-6-interacting corepressor) or its paralog, BCOR-like 1 (BCORL1). Previous structural studies revealed that the C-terminal PUFD domain of BCORL1 is necessary and sufficient to heterodimerize with the RAWUL domain of PCGF1 and, together, form a new protein-protein binding interface that associates with the histone demethylase KDM2B. Here, we show that the PUFD of BCOR and BCORL1 differ in their abilities to assemble with KDM2B. Unlike BCORL1, the PUFD of BCOR alone does not stably assemble with KDM2B. Rather, additional residues N-terminal to the BCOR PUFD are necessary for stable association. Nuclear magnetic resonance (NMR) structure determination and N relaxation time measurements of the BCOR PUFD alone indicate that the termini of the BCOR PUFD, which are critical for binding PCGF1 and KDM2B, are disordered. This suggests a hierarchical mode of assembly whereby BCOR PUFD termini become structurally ordered upon binding PCGF1, which then allows stable association with KDM2B. Notably, internal tandem duplications (ITDs) leading to pediatric kidney and brain tumors map to the PUFD termini. Binding studies with the BCOR ITD indicate the ITD would disrupt PRC1.1 assembly, suggesting loss of the ability to assemble PRC1.1 is a critical molecular event driving tumorigenesis.
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http://dx.doi.org/10.1021/acs.biochem.0c00285DOI Listing
July 2020

Structural biology of betaglycan and endoglin, membrane-bound co-receptors of the TGF-beta family.

Exp Biol Med (Maywood) 2019 12 10;244(17):1547-1558. Epub 2019 Oct 10.

Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA.

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http://dx.doi.org/10.1177/1535370219881160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920675PMC
December 2019

Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin.

Structure 2019 09 18;27(9):1427-1442.e4. Epub 2019 Jul 18.

Department of Structural Biology, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, Room 2051, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA. Electronic address:

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-β family, potentiate the signaling activity of TGF-β2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BG) reveals an insertion that blocks the region that the endoglin orphan domain (ENG) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-β and BG, as well as small-angle X-ray scattering data, BG is shown to bind its cognate GF in an entirely different manner compared with ENG. The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-β family GFs.
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http://dx.doi.org/10.1016/j.str.2019.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726503PMC
September 2019

Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity.

Proc Natl Acad Sci U S A 2019 07 17;116(31):15505-15513. Epub 2019 Jul 17.

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267;

TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.
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http://dx.doi.org/10.1073/pnas.1906253116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681762PMC
July 2019

TGF-β2 uses the concave surface of its extended finger region to bind betaglycan's ZP domain via three residues specific to TGF-β and inhibin-α.

J Biol Chem 2019 03 31;294(9):3065-3080. Epub 2018 Dec 31.

From the Departments of Structural Biology and

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-β family that selectively binds transforming growth factor-β (TGF-β) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions Here, using NMR titrations of methyl-labeled TGF-β2 with BG's C-terminal binding domain, BG, and surface plasmon resonance binding measurements with TGF-β2 variants, we found that the BG-binding site on TGF-β2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-β isoforms and the InhA α-subunit, but they are unconserved in other TGF-β family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-β2 variants with the corresponding residues from BMP-2 bound BG more weakly than corresponding alanine variants. The BG-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-β2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-β2 and InhA bind BG through a site on the inside of their extended finger regions. Overall, these results identify the BG-binding site on TGF-β2 and shed light on the specificity of BG for select TGF-β-type GFs and the mechanisms by which BG influences their signaling.
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http://dx.doi.org/10.1074/jbc.RA118.005210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398128PMC
March 2019

Structure-guided engineering of TGF-βs for the development of novel inhibitors and probing mechanism.

Authors:
Andrew P Hinck

Bioorg Med Chem 2018 10 7;26(19):5239-5246. Epub 2018 Jul 7.

Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address:

The increasing availability of detailed structural information on many biological systems provides an avenue for manipulation of these structures, either for probing mechanism or for developing novel therapeutic agents for treating disease. This has been accompanied by the advent of several powerful new methods, such as the ability to incorporate non-natural amino acids or perform fragment screening, increasing the capacity to leverage this new structural information to aid in these pursuits. The abundance of structural information also provides new opportunities for protein engineering, which may become more and more relevant as treatment of diseases using gene therapy approaches become increasingly common. This is illustrated by example with the TGF-β family of proteins, for which there is ample structural information, yet no approved inhibitors for treating diseases, such as cancer and fibrosis that are promoted by excessive TGF-β signaling. The results presented demonstrate that through several relatively simple modifications, primarily involving the removal of an α-helix and replacement of it with a flexible loop, it is possible to alter TGF-βs from being potent signaling proteins into inhibitors of TGF-β signaling. The engineered TGF-βs have improved specificity relative to kinase inhibitors and a much smaller size compared to monoclonal antibodies, and thus may prove successful as either as an injected therapeutic or as a gene therapy-based therapeutic, where other classes of inhibitors have failed.
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http://dx.doi.org/10.1016/j.bmc.2018.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797196PMC
October 2018

A Novel TGFβ Trap Blocks Chemotherapeutics-Induced TGFβ1 Signaling and Enhances Their Anticancer Activity in Gynecologic Cancers.

Clin Cancer Res 2018 06 16;24(12):2780-2793. Epub 2018 Mar 16.

Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

We investigated the mechanisms of how TGFβ pathway is activated by chemotherapeutics and whether a novel TGFβ trap called RER can block chemotherapeutics-induced TGFβ pathway activation and enhance their antitumor activity in gynecologic cancer. An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFβ signaling and function, and the effects of RER on chemotherapy-induced TGFβ signaling were determined in gynecologic cancer cells. Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFβ signaling activation. Chemotherapy was found to activate TGFβ signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFβ1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFβ-like effects were due to the stimulation of TGFβ1 expression and secretion, and could all be abrogated by TGFβ inhibitors including a novel TGFβ trap protein called RER both and Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer. Chemotherapeutics can stimulate TGFβ1 production and consequently enhance TGFβ signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFβ inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004245PMC
June 2018

TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition.

Elife 2018 01 29;7. Epub 2018 Jan 29.

Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom.

The best characterized signaling pathway downstream of transforming growth factor β (TGF-β) is through SMAD2 and SMAD3. However, TGF-β also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-β-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the genes. Finally, we show that TGF-β-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-β-induced transcriptional program and physiological responses.
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http://dx.doi.org/10.7554/eLife.31756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832415PMC
January 2018

A structurally distinct TGF-β mimic from an intestinal helminth parasite potently induces regulatory T cells.

Nat Commun 2017 11 23;8(1):1741. Epub 2017 Nov 23.

Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-β signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing mouse and human Foxp3 Treg cells. Hp-TGM has no homology with mammalian TGF-β or other members of the TGF-β family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.
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http://dx.doi.org/10.1038/s41467-017-01886-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701006PMC
November 2017

ALK1 signaling in development and disease: new paradigms.

Cell Mol Life Sci 2017 12 4;74(24):4539-4560. Epub 2017 Sep 4.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Activin A receptor like type 1 (ALK1) is a transmembrane serine/threonine receptor kinase in the transforming growth factor-beta receptor family that is expressed on endothelial cells. Defects in ALK1 signaling cause the autosomal dominant vascular disorder, hereditary hemorrhagic telangiectasia (HHT), which is characterized by development of direct connections between arteries and veins, or arteriovenous malformations (AVMs). Although previous studies have implicated ALK1 in various aspects of sprouting angiogenesis, including tip/stalk cell selection, migration, and proliferation, recent work suggests an intriguing role for ALK1 in transducing a flow-based signal that governs directed endothelial cell migration within patent, perfused vessels. In this review, we present an updated view of the mechanism of ALK1 signaling, put forth a unified hypothesis to explain the cellular missteps that lead to AVMs associated with ALK1 deficiency, and discuss emerging roles for ALK1 signaling in diseases beyond HHT.
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http://dx.doi.org/10.1007/s00018-017-2636-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687069PMC
December 2017

An introduction to the special issue on biomolecular NMR.

Arch Biochem Biophys 2017 08 27;628:1-2. Epub 2017 Jun 27.

Instituto de Biología Molecular y Celular, Universidad Miguel Hernández de Elche, Elche, Alicante 03202, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.abb.2017.06.020DOI Listing
August 2017

Structural basis for potency differences between GDF8 and GDF11.

BMC Biol 2017 03 3;15(1):19. Epub 2017 Mar 3.

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH, 45267, USA.

Background: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties.

Results: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8.

Conclusions: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined.
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http://dx.doi.org/10.1186/s12915-017-0350-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336696PMC
March 2017

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

J Biol Chem 2017 04 22;292(17):7173-7188. Epub 2017 Feb 22.

From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260,

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 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.
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http://dx.doi.org/10.1074/jbc.M116.768754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409485PMC
April 2017

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.

Nat Methods 2017 Feb;14(4):399-402

Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
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http://dx.doi.org/10.1038/nmeth.4178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376236PMC
February 2017

Binding Properties of the Transforming Growth Factor-β Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling.

Biochemistry 2016 12 2;55(49):6880-6896. Epub 2016 Dec 2.

Department of Structural Biology, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1-TGF-β3, respectively) are small secreted signaling proteins that 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.
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http://dx.doi.org/10.1021/acs.biochem.6b00566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551644PMC
December 2016

A novel highly potent trivalent TGF-β receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

Oncotarget 2016 Dec;7(52):86087-86102

Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA.

The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.
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http://dx.doi.org/10.18632/oncotarget.13343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349899PMC
December 2016

Structural Biology and Evolution of the TGF-β Family.

Cold Spring Harb Perspect Biol 2016 Dec 1;8(12). Epub 2016 Dec 1.

Program in Cellular and Molecular Medicine and Division of Hematology, Department of Medicine, Boston Children's Hospital, Boston, Massachusetts 02115.

We review the evolution and structure of members of the transforming growth factor β (TGF-β) family, antagonistic or agonistic modulators, and receptors that regulate TGF-β signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazoans, including the TGF-β and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-β family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-β family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex conformations. Ternary complexes of the final step in extracellular signaling show how TGF-β GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo.
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http://dx.doi.org/10.1101/cshperspect.a022103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131774PMC
December 2016

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily.

Methods Mol Biol 2016 ;1344:63-92

Protein Chemistry, Novo Nordisk Research Center China, 20 Life Science Park Rd, Bldg 2, Beijing, 102206, China.

The ability to understand the molecular mechanisms by which secreted signaling proteins of the TGF-β superfamily assemble their cell surface receptors into complexes to initiate downstream signaling is dependent upon the ability to determine atomic-resolution structures of the signaling proteins, the ectodomains of the receptors, and the complexes that they form. The structures determined to date have revealed major differences in the overall architecture of the signaling complexes formed by the TGF-βs and BMPs, which has provided insights as to how they have evolved to fulfill their distinct functions. Such studies, have however, only been applied to a few members of the TGF-β superfamily, which is largely due to the difficulty of obtaining milligram-scale quantities of highly homogenous preparations of the disulfide-rich signaling proteins and receptor ectodomains of the superfamily. Here we describe methods used to produce signaling proteins and receptor ectodomains of the TGF-β superfamily using bacterial and mammalian expression systems and procedures to purify them to homogeneity.
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http://dx.doi.org/10.1007/978-1-4939-2966-5_4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846357PMC
May 2016

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

Biochemistry 2015 Aug 13;54(33):5167-74. Epub 2015 Aug 13.

Department of Biochemistry, University of Texas Health Science Center , San Antonio, Texas 78229, United States.

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.
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http://dx.doi.org/10.1021/acs.biochem.5b00616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551101PMC
August 2015

Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain.

Biochemistry 2015 Apr 16;54(16):2571-80. Epub 2015 Apr 16.

Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.

The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of (13)C-, (2)H-, and (15)N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event.
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http://dx.doi.org/10.1021/acs.biochem.5b00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429812PMC
April 2015

Divergence(s) in nodal signaling between aggressive melanoma and embryonic stem cells.

Int J Cancer 2015 Mar 29;136(5):E242-51. Epub 2014 Sep 29.

Cancer Biology and Epigenomics Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL.

The significant role of the embryonic morphogen Nodal in maintaining the pluripotency of embryonic stem cells is well documented. Interestingly, the recent discovery of Nodal's re-expression in several aggressive and metastatic cancers has highlighted its critical role in self renewal and maintenance of the stem cell-like characteristics of tumor cells, such as melanoma. However, the key TGFβ/Nodal signaling component(s) governing Nodal's effects in metastatic melanoma remain mostly unknown. By employing receptor profiling at the mRNA and protein level(s), we made the novel discovery that embryonic stem cells and metastatic melanoma cells share a similar repertoire of Type I serine/threonine kinase receptors, but diverge in their Type II receptor expression. Ligand:receptor crosslinking and native gel binding assays indicate that metastatic melanoma cells employ the heterodimeric TGFβ receptor I/TGFβ receptor II (TGFβRI/TGFβRII) for signal transduction, whereas embryonic stem cells use the Activin receptors I and II (ACTRI/ACTRII). This unexpected receptor usage by tumor cells was tested by: neutralizing antibody to block its function; and transfecting the dominant negative receptor to compete with the endogenous receptor for ligand binding. Furthermore, a direct biological role for TGFβRII was found to underlie vasculogenic mimicry (VM), an endothelial phenotype contributing to vascular perfusion and associated with the functional plasticity of aggressive melanoma. Collectively, these findings reveal the divergence in Nodal signaling between embryonic stem cells and metastatic melanoma that can impact new therapeutic strategies targeting the re-emergence of embryonic pathways.
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http://dx.doi.org/10.1002/ijc.29198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465342PMC
March 2015

Biological activity differences between TGF-β1 and TGF-β3 correlate with differences in the rigidity and arrangement of their component monomers.

Biochemistry 2014 Sep 5;53(36):5737-49. Epub 2014 Sep 5.

Department of Biochemistry, University of Texas Health Science Center at San Antonio , San Antonio, Texas 78229-3900, United States.

TGF-β1, -β2, and -β3 are small, secreted signaling proteins. They 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. Surface plasmon resonance analysis showed that TGF-β1, TGF-β3, and variants bound the type II signaling receptor, TβRII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling receptor, TβRI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that differentially binds them might determine their distinct activities.
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http://dx.doi.org/10.1021/bi500647dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165442PMC
September 2014

The solution structure of the regulatory domain of tyrosine hydroxylase.

J Mol Biol 2014 Apr 17;426(7):1483-97. Epub 2013 Dec 17.

Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1-71) and a well-folded C-terminal portion (residues 72-159). The structure of a truncated version of the regulatory domain containing residues 65-159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65-159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH.
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http://dx.doi.org/10.1016/j.jmb.2013.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951675PMC
April 2014

TGF-β antagonists: same knot, but different hold.

Structure 2013 Aug;21(8):1269-70

Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA.

In this issue of Structure, Nolan and colleagues present the structure of BMP antagonist, PRDC, which adopts a head-to-tail dimer with distinct structure and inhibitory mechanism compared to other dimeric antagonists of the TGF-β superfamily, such as noggin.
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http://dx.doi.org/10.1016/j.str.2013.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809007PMC
August 2013

Blockade of Autocrine TGF-β Signaling Inhibits Stem Cell Phenotype, Survival, and Metastasis of Murine Breast Cancer Cells.

J Stem Cell Res Ther 2012 Feb;2(1):1-8

Department of Cellular & Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229, USA ; Department of Breast Surgery, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, 210029 Nanjing, China.

Transforming growth factor beta (TGF-β) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study, we have utilized a novel murine cell line NMuMG-ST, which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG, to investigate the role of autocrine TGF-β signaling in regulating their survival, metastatic ability, and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-β type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-β signaling. The expression of DNRII reduced TGF-β sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-β signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK, and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly, the abrogation of autocrine TGF-β signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition, mammosphere formation, and expression of stem cell markers. When xenografted in athymic nude mice, the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus, our results indicate that autocrine TGF-β signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells.
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http://dx.doi.org/10.4172/2157-7633.1000116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593047PMC
February 2012

Structure of the Alk1 extracellular domain and characterization of its bone morphogenetic protein (BMP) binding properties.

Biochemistry 2012 Aug 2;51(32):6328-41. Epub 2012 Aug 2.

Department of Biochemistry and Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA.

Bone morphogenetic proteins (BMPs) are secreted signaling proteins - they transduce their signals by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homologue Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the β-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the β4-β5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the β4-β5 loop, and to lesser extent β1-β2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9/Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3 but is rotated by 40 deg. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the β4-β5 loop that contribute significantly to binding and are nonconservatively substituted relative to the corresponding residues in Alk3.
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http://dx.doi.org/10.1021/bi300942xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448977PMC
August 2012