Signal processing in the TGF-beta superfamily ligand-receptor network

The TGF-beta pathway plays a central role in tissue homeostasis and morphogenesis. It transduces a variety of extracellular signals into intracellular transcriptional responses that control a plethora of cellular processes, including cell growth, apoptosis, and differentiation. We use computational modeling to show that coupling of signaling with receptor trafficking results in a highly versatile signal-processing unit, able to sense by itself absolute levels of ligand, temporal changes in ligand concentration, and ratios of multiple ligands. This coupling controls whether the response of the receptor module is transient or permanent and whether or not different signaling channels behave independently of each other. Our computational approach unifies seemingly disparate experimental observations and suggests specific changes in receptor trafficking patterns that can lead to phenotypes that favor tumor progression.Comment: 33 pages, 7 figure

Trafficking Coordinate Description of Intracellular Transport Control of Signaling Networks

Many cellular networks rely on the regulated transport of their components to transduce extracellular information into precise intracellular signals. The dynamics of these networks is typically described in terms of compartmentalized chemical reactions. There are many important situations, however, in which the properties of the compartments change continuously in a way that cannot naturally be described by chemical reactions. Here, we develop an approach based on transport along a trafficking coordinate to precisely describe these processes and we apply it explicitly to the TGF-{\beta} signal transduction network, which plays a fundamental role in many diseases and cellular processes. The results of this newly introduced approach accurately capture for the first time the distinct TGF-{\beta} signaling dynamics of cells with and without cancerous backgrounds and provide an avenue to predict the effects of chemical perturbations in a way that closely recapitulates the observed cellular behavior.Comment: 17 pages, 5 figure

SARS-CoV Regulates Immune Function-Related Gene Expressions in Human Monocytic Cells

Background: Severe Acute Respiratory Syndrome (SARS) is characterized by acute respiratory distress (ARDS) and pulmonary fibrosis, and the monocyte/macrophage is the key player in the pathogenesis of SARS.
 
Methods: In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV) infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection and the gene expression was profiled by oligonucleotide-based microarray. 

Results: Analysis of immune-related gene expression profiles showed that 24 h after SARS-CoV infection, (i) IFN-alpha/beta-inducible and cathepsin/proteosome genes were down-regulated; (ii) the hypoxia/hyperoxia-related genes were up-regulated; and (iii) the TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, the lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. 

Conclusion: These results elucidate that monocyte/macrophage dysfunction and dysregulation of fibrosis-related genes are two important pathogenic events of SARS. 
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A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor

BACKGROUND: Bone morphogenetic proteins (BMPs) are key regulators in the embryonic development and postnatal tissue homeostasis in all animals. Loss of function or dysregulation of BMPs results in severe diseases or even lethality. Like transforming growth factors β (TGF-βs), activins, growth and differentiation factors (GDFs) and other members of the TGF-β superfamily, BMPs signal by assembling two types of serine/threonine-kinase receptor chains to form a hetero-oligomeric ligand-receptor complex. BMP ligand receptor interaction is highly promiscuous, i.e. BMPs bind more than one receptor of each subtype, and a receptor bind various ligands. The activin type II receptors are of particular interest, since they bind a large number of diverse ligands. In addition they act as high-affinity receptors for activins but are also low-affinity receptors for BMPs. ActR-II and ActR-IIB therefore represent an interesting example how affinity and specificity might be generated in a promiscuous background. RESULTS: Here we present the high-resolution structures of the ternary complexes of wildtype and a variant BMP-2 bound to its high-affinity type I receptor BMPR-IA and its low-affinity type II receptor ActR-IIB and compare them with the known structures of binary and ternary ligand-receptor complexes of BMP-2. In contrast to activin or TGF-β3 no changes in the dimer architecture of the BMP-2 ligand occur upon complex formation. Functional analysis of the ActR-IIB binding epitope shows that hydrophobic interactions dominate in low-affinity binding of BMPs; polar interactions contribute only little to binding affinity. However, a conserved H-bond in the center of the type II ligand-receptor interface, which does not contribute to binding in the BMP-2 – ActR-IIB interaction can be mutationally activated resulting in a BMP-2 variant with high-affinity for ActR-IIB. Further mutagenesis studies were performed to elucidate the binding mechanism allowing us to construct BMP-2 variants with defined type II receptor binding properties. CONCLUSION: Binding specificity of BMP-2 for its three type II receptors BMPR-II, Act-RII and ActR-IIB is encoded on single amino acid level. Exchange of only one or two residues results in BMP-2 variants with a dramatically altered type II receptor specificity profile, possibly allowing construction of BMP-2 variants that address a single type II receptor. The structure-/function studies presented here revealed a new mechanism, in which the energy contribution of a conserved H-bond is modulated by surrounding intramolecular interactions to achieve a switch between low- and high-affinity binding

Developing Chemical Tools for the Inhibition of Activin A Signalling

Activin A is a member of the TGF-β superfamily, a family of structurally related growth factors that have been implicated in embryogenesis, homeostasis, and cancer. All members consist of pro- and mature domains which are post translationally cleaved. Upon signalling the pro-domain dissociates from the mature growth factor. Signalling in this family requires a type I and a type II receptor. Though there are over thirty growth factors, there are only seven type I and five type II receptors resulting in promiscuity between ligands and receptors. In order to fully elucidate the roles of these proteins and develop therapeutics, specific inhibitors must be generated. In this thesis, I targeted activin A to achieve specificity. I first targeted mature activin A through the use of XChem, conducting a fragment screen against mature activin A crystals. I describe the identification of twelve fragment hits that bind in the putative type I receptor binding site and the validation and optimisation of one of these fragments. I then used the structure of the pro-mature complex of activin A to design and generate a series of GB1- fused peptides based on the α1-helix-loop-α2-helix motif of the pro-domain. I determined the α1-helix-loop epitope to be key for interactions with the mature domain, before investigating the effect of truncation and mutation on this epitope. I then determined the potency of the fusion GB1-H1LH2 as an inhibitor of activin A signalling (IC50 = 4.4 μM). I further optimised GB1-H1LH2 through dimerization resulting in a large increase in inhibitor potency (IC50 = 71.5 nM). Lastly, I screened this dimeric inhibitor against several TGF-β family members to determine it specifically inhibits both activin A and activin B. To conclude, I discuss the findings of this thesis before considering how this methodology could be applied to other TGF-β family members

Transforming growth factor-β superfamily, implications in development and differentiation of stem cells

Transforming growth factor-β (TGF-β) family members, including TGF-βs and bone morphogenetic proteins (BMPs), play important roles in directing the fate of stem cells. In embryonic stem cells, the TGF-β superfamily participates in almost all stages of cell development, such as cell maintenance, lineage selection, and progression of differentiation. In adult mesenchymal stem cells (MSCs), TGF-βs can provide competence for early stages of chondroblastic and osteoblastic differentiation, but they inhibit myogenesis, adipogenesis, and late-stage osteoblast differentiation. BMPs also inhibit adipogenesis and myogenesis, but they strongly promote osteoblast differentiation. The TGF-β superfamily members signal via specific serine/threonine kinase receptors and their nuclear effectors termed Smad proteins as well as through non-Smad pathways, which explain their pleiotropic effects in self-renewal and differentiation of stem cells. This review summarizes the current knowledge on the pleiotropic effects of the TGF-β superfamily of growth factors on the fate of stem cells and also discusses the mechanisms by which the TGF-β superfamily members control embryonic and MSCs differentiation

Alk1 Signaling in Vascular Development

Heterozygous loss of the endothelial-specific transforming growth factor-beta (TGF-β) Type 1 receptor, activin receptor-like kinase 1 (ALK1), results in the autosomal dominant disorder, hereditary hemorrhagic telangiectasia type 2 (HHT2), which is characterized by mucocutaneous telangiectasias as well as arteriovenous malformations (AVMs) in the brain, lungs, liver, gastrointestinal tract, and spinal cord. As a result, patients suffer from a range of clinical symptoms including epistaxis, hemorrhage, and stroke. Using zebrafish, our laboratory has demonstrated that AVMs form via a two-step mechanism involving an initial increase in endothelial cell number caused by lack of alk1, and then an adaptive response to increased blood flow in downstream vessels. This adaptive response involves increased arterial caliber and maintenance of normally transient connections between arteries and veins, thereby forming high-flow AVMs. Furthermore, we have demonstrated that alk1 expression is dependent on blood flow, and that lack of flow mimics loss of alk1, suggesting that Alk1 might act downstream of blood flow to stabilize arterial caliber. To date, the in vivo ligand and intracellular mediators required for flow-dependent, Alk1-mediated endothelial quiescence and AVM prevention remain unknown. In this work, I demonstrate that bone morphogenetic protein 10 (Bmp10) is the physiologically relevant Alk1 ligand during zebrafish embryonic development. Bmp10 paralogs are expressed exclusively in the heart, and loss of blood flow affects arterial pSmad1/5/9, cxcr4a, and edn1 expression similarly to loss of alk1, even when alk1 expression is restored via a flow-independent transgene. Together, these data suggest that flow is required not only for alk1 expression but also to deliver cardiac-derived Bmp10 ligand to arterial endothelial cell Alk1 to promote endothelial cell quiescence. Downstream of Bmp10/Alk1, Alk1 kinase activity is required to prevent AVMs. However characterization of a pSmad1/5-responsive transgenic reporter, Tg(BRE:EGFP), suggests that although phosphorylation of Smad1/5/9 in arterial endothelium is clearly dependent on Alk1, pSmad1/5/9 may not activate transcription via a canonical mechanism within these cells. In sum, the work presented in this thesis constructs a novel blood flow-responsive signaling pathway, suggests novel mechanisms by which Alk1 may control gene expression, and finally, describes a new tool for studying Alk1 and BMP signaling in vivo