Heparin, Heparan Sulphate and the TGF-ß Cytokine Superfamily.

Of the circa 40 cytokines of the TGF-ß superfamily, around a third are currently known to bind to heparin and heparan sulphate. This includes TGF-ß1, TGF-ß2, certain bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), as well as GDNF and two of its close homologues. Experimental studies of their heparin/HS binding sites reveal a diversity of locations around the shared cystine-knot protein fold. The activities of the TGF-ß cytokines in controlling proliferation, differentiation and survival in a range of cell types are in part regulated by a number of specific, secreted BMP antagonist proteins. These vary in structure but seven belong to the CAN or DAN family, which shares the TGF-ß type cystine-knot domain. Other antagonists are more distant members of the TGF-ß superfamily. It is emerging that the majority, but not all, of the antagonists are also heparin binding proteins. Any future exploitation of the TGF-ß cytokines in the therapy of chronic diseases will need to fully consider their interactions with glycosaminoglycans and the implications of this in terms of their bioavailability and biological activity.

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability.

The specific functions of the prodomains of TGFß superfamily members are largely unknown. Interactions are known between prodomains of TGFß-1-3 and latent TGFß-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFß-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFß superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFß and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.

Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-ß Family.

The 33 members of the transforming growth factor beta (TGF-ß) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-ß1 and TGF-ß2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-ß family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.

Prodomains regulate the synthesis, extracellular localisation and activity of TGF-ß superfamily ligands.

All transforming growth factor-ß (TGF-ß) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-ß ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-ß ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-ß1, TGF-ß2, TGF-ß3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-ß ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-ß superfamily biology, and alterations in prodomain function are often associated with disease.

The three-fingered protein domain of the human genome.

Extracellular domains of some cellular receptors expressed in the organisms at different levels of development belong to three-fingered protein (TFP) fold. The Homo sapiens genome encodes at least 45 genes containing from one to three TFP domains (TFPDs), namely diverse paralogues of the Ly6 gene, CD59 and the receptors of activins, bone morphogenetic proteins, Mullerian inhibiting substance and transforming growth factor-beta. C4.4a and urokinase/plasminogen activatory receptor contain two and three TFPD repeats, respectively. These diverse proteins have a low overall sequence similarity with each other and their hydrophobicity levels vary to a considerable degree. It is suggested that sequence differentiation within the TFPD led to distinct groups of proteins whose attributes were optimized to fit both the physicochemical properties specific to their functional microenvironment and selective targeting of their highly diversified extracellular cofactors.

Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-ß family.

The kinetics of folding and dimerization of bone morphogenetic protein-2 (BMP-2), a disulfide-connected, homodimeric cystine-knot protein and a member of the transforming growth factor-ß superfamily, was analyzed under a variety of different conditions. Refolding and dimerization of BMP-2 were extremely slow under all conditions studied, and could be described by consecutive first-order reactions involving at least one long-lived intermediate. The rate constants vary from ~ 0.2 × 10(-5) to ~ 3.5 × 10(-5) s(-1), and were strongly dependent on temperature, redox conditions, and the presence of stabilizing or destabilizing ions. In particular, the combined impact of ionic strength and redox conditions on the rates indicates that electrostatic interactions control thiol-disulfide exchange reactions on the path from the unfolded and reduced monomers to the disulfide-connected growth factor in a rate-determining way.

Toward a better understanding of the interaction between TGF-ß family members and their ALK receptors.

Transforming growth factor-beta (TGF-ß) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-ß/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-ß ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-ß/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not.

Intricacies of BMP receptor assembly.

The TGF-beta superfamily exhibits a feature making it distinct from many other growth factor families in that the inadequate number of ligands and receptors premises a high degree of promiscuity in ligand-receptor interaction. This highlights the importance of even small differences in affinities and specificities between different binding partners to maintain the broad spectrum of their well defined biological functions. Despite the promiscuous interactions recent data reveal differences in receptor recruitment, architectures of these assemblies and specific modulation by a multitude of extracellular as well as membrane-associated factors. These modulatory mechanisms might possibly add specificity towards defined biological functions despite the overlapping usage of receptors by various ligands.