Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-ß responses through SKI stabilization.

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-ß signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-ß signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-ß responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-ß-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

Calreticulin is a secreted BMP antagonist, expressed in Hensen's node during neural induction.

Hensen's node is the "organizer" of the avian and mammalian early embryo. It has many functions, including neural induction and patterning of the ectoderm and mesoderm. Some of the signals responsible for these activities are known but these do not explain the full complexity of organizer activity. Here we undertake a functional screen to discover new secreted factors expressed by the node at this time of development. Using a Signal Sequence Trap in yeast, we identify several candidates. Here we focus on Calreticulin. We show that in addition to its known functions in intracellular Calcium regulation and protein folding, Calreticulin is secreted, it can bind to BMP4 and act as a BMP antagonist in vivo and in vitro. Calreticulin is not sufficient to account for all organizer functions but may contribute to the complexity of its activity.

Conformational dependence of through-space tellurium-tellurium spin-spin coupling in peri-substituted bis(tellurides).

Three related series of peri-substituted bis(tellurides) bearing naphthalene, acenaphthene and acenaphthylene backbones (Nap/Acenap/Aceyl(TeY)2 (Nap = naphthalene-1,8-diyl N; Acenap = acenaphthene-5,6-diyl A; Aceyl = acenaphthylene-5,6-diyl Ay; Y = Ph 1; Fp 2; Tol 3; An-p- 4; An-o- 5; Tp 6; Mes 7; Tip 8) have been synthesised and their solid-state structures determined by X-ray crystallography. Molecular conformations were classified as a function of the two C9-C-Te-C(Y) dihedral angles (θ); in the solid all members adopt AB or CCt configurations, with larger Te(aryl) moieties exclusively imposing the CCt variant. Exceptionally large J((125)Te,(125)Te) spin-spin coupling constants between 3289-3848 Hz were obtained for compounds substituted by bulky Te(aryl) groups, implying these species are locked in a CCt-type conformation. In contrast, compounds incorporating smaller Te(aryl) moieties are predicted to be rather dynamic in solution and afford much smaller J values (2050-2676 Hz), characteristic of greater populations of AB conformers with lower couplings. This conformational dependence of through-space coupling is supported by DFT calculations.

A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border.

During ectodermal patterning the neural crest and preplacodal ectoderm are specified in adjacent domains at the neural plate border. BMP signalling is required for specification of both tissues, but how it is spatially and temporally regulated to achieve this is not understood. Here, using a transgenic zebrafish BMP reporter line in conjunction with double-fluorescent in situ hybridisation, we show that, at the beginning of neurulation, the ventral-to-dorsal gradient of BMP activity evolves into two distinct domains at the neural plate border: one coinciding with the neural crest and the other abutting the epidermis. In between is a region devoid of BMP activity, which is specified as the preplacodal ectoderm. We identify the ligands required for these domains of BMP activity. We show that the BMP-interacting protein Crossveinless 2 is expressed in the BMP activity domains and is under the control of BMP signalling. We establish that Crossveinless 2 functions at this time in a positive-feedback loop to locally enhance BMP activity, and show that it is required for neural crest fate. We further demonstrate that the Distal-less transcription factors Dlx3b and Dlx4b, which are expressed in the preplacodal ectoderm, are required for the expression of a cell-autonomous BMP inhibitor, Bambi-b, which can explain the specific absence of BMP activity in the preplacodal ectoderm. Taken together, our data define a BMP regulatory network that controls cell fate decisions at the neural plate border.

Electrochemically informed synthesis: oxidation versus coordination of 5,6-bis(phenylchalcogeno)acenaphthenes.

Chalcogen dications: Facile synthesis of E--E bonded dications can be readily achieved. Radical cations are identified as the intermediates.

Synthetic, structural, NMR, and computational study of a geminally bis(peri-substituted) tridentate phosphine and its chalcogenides and transition-metal complexes.

Coupling of two acenaphthene backbones through a phosphorus atom in a geminal fashion gives the first geminally bis(peri-substituted) tridentate phosphine 1. The rigid nature of the aromatic backbone and overall crowding of the molecule result in a rather inflexible ligand, with the three phosphorus atoms forming a relatively compact triangular cluster. Phosphine 1 displays restricted dynamics on an NMR time scale, which leads to the anisochronicity of all three phosphorus nuclei at low temperatures. Strained bis- and tris(sulfides) 2 and 3 and the bis(selenide) 4 have been isolated from the reaction of 1 with sulfur and selenium, respectively. These chalcogeno derivatives display pronounced in-plane and out-of-plane distortions of the aromatic backbones, indicating the limits of their angular distortions. In addition, we report metal complexes with tetrahedral [(1)Cu(MeCN)][BF4] (5), square planar [(1)PtCl][Cl] (6), trigonal bipyramidal [(1)FeCl2] (7), and octahedral fac-[(1)Mo(CO)3] (8) geometries. In all of these complexes the tris(phosphine) backbone is distorted, however to a significantly smaller extent than that in the mentioned chalcogenides 2-4. Complexes 5 and 8 show fluxionality in (31)P and (1)H NMR. All new compounds 1-8 were fully characterized, and their crystal structures are reported. Conclusions from dynamic NMR observations were augmented by DFT calculations.

An efficient route for the synthesis of phosphorus-selenium macro-heterocycles.

Four-membered ring [PhP(Se)(µ-Se)]2 (Woollins' reagent, WR) reacts with disodium alkenyl-diols followed by in situ ring-closure reaction with appropriate dibromoalkanes affording a series of unusual nine- to fifteen-membered organoselenophosphorus macrocycles bearing the O-P-Se-Cn-Se-P-O or O-P-Se-Cn-O-P-Se linkage.

Weak Te,Te interactions through the looking glass of NMR spin-spin coupling.

Across the bay: J((125)Te, (125)Te) spin-spin coupling is a highly sensitive probe into the electronic and geometric structure of 1,8-peri-substituted naphthalene tellurium derivatives. The coupling is related to the onset of multicenter bonding in these systems.

Silver(I) coordination complexes and extended networks assembled from S, Se, Te substituted acenaphthenes.

Six related organo­chalconium silver(I) coordination complexes, including two examples of rare organotellurium-silver coordination, have been prepared and structurally characterised by X-ray crystallography. The series of 5-bromo-6-(phenylchalcogeno)acenaphthene ligands L1­L3 [Acenap(Br)(EPh)] (Acenap = acenaphthene-5,6-diyl; E = S, Se, Te) were independently treated with silver(I) salts (AgBF4, AgOTf). In order to keep the number of variables to a minimum, all reactions were carried out using a 1:1 ratio of Ag/L and run in dichloromethane. The nature of the donor atoms and the coordinating ability of the respective counter-anion affects the structural architecture of the final silver(I) complex, generating a monomeric dinuclear complex {[(AgBF4(L1)2)2] 1}, monomeric, mononuclear, two-coordinate silver(I) complexes {[AgBF4(L)2] (2 L = L2; 3 L = L3)}, a monomeric three-coordinate silver(I) complex {[AgOTf(L2)2] 5}, a monomeric four-coordinate silver(I) complex {[AgOTf(L1)3] 4} and a 1D extended helical chain polymer {[AgOTf(L3)]n 6}. The organic acenaphthene ligands L1­L3 all adopt the same ligation mode with the central silver atom (classical monodentate coordination), which employs a variety of coordination geometries (linear, trigonal planar, see-saw, tetrahedral).

Investigating silver coordination to mixed chalcogen ligands.

Six silver(I) coordination complexes have been prepared and structurally characterised. Mixed chalcogen-donor acenaphthene ligands L1-L3 [Acenap(EPh)(E'Ph)] (Acenap = acenaphthene-5,6-diyl; E/E' = S, Se, Te) were independently treated with silver(I) salts (AgBF4/AgOTf). In order to keep the number of variables to a minimum, all reactions were carried out using a 1:1 ratio of Ag/L and run in dichloromethane. The nature of the donor atoms, the coordinating ability of the respective counter-anion and the type of solvent used in recrystallisation, all affect the structural architecture of the final silver(I) complex, generating monomeric, silver(I) complexes {[AgBF4(L)2] (1 L = L1; 2 L = L2; 3 L = L3), [AgOTf(L)3] (4 L = L1; 5 L = L3), [AgBF4(L)3] (2a L = L1; 3a L = L3)} and a 1D polymeric chain {[AgOTf(L3)](n) 6}. The organic acenaphthene ligands L1-L3 adopt a number of ligation modes (bis-monodentate µ2-η²-bridging, quasi-chelating combining monodentate and η6-E(phenyl)-Ag(I) and classical monodentate coordination) with the central silver atom at the centre of a tetrahedral or trigonal planar coordination geometry in each case. The importance of weak interactions in the formation of metal-organic structures is also highlighted by the number of short non-covalent contacts present within each complex.

Noncovalent interactions in peri-substituted chalconium acenaphthene and naphthalene salts: a combined experimental, crystallographic, computational, and solid-state NMR study.

Twelve related monocation chalconium salts [{Nap(EPh)(E'Ph)Me}(+){CF(3)SO(3)}(-)] 2-4, [{Acenap(Br)(EPh)Me}{CF(3)SO(3)}(-)] 5-7, and [{Acenap(EPh)(E'Ph)Me}(+){CF(3)SO(3)}(-)] 8-13 have been prepared and structurally characterized. For their synthesis naphthalene compounds [Nap(EPh)(E'Ph)] (Nap = naphthalene-1,8-diyl; E/E' = S, Se, Te) N2-N4 and associated acenaphthene derivatives [Acenap(X)(EPh)]/[Acenap(EPh)(E'Ph)] (Acenap = acenaphthene-5,6-diyl; E/E' = S, Se, Te; X = Br) A5-A13 were independently treated with a single molar equivalent of methyl trifluoromethanesulfonate (MeOTf). In addition, reaction of bis-tellurium compound A10 with 2 equiv of MeOTf afforded the doubly methylated dication salt [{Acenap(TePhMe)(2)}(2+){(CF(3)SO(3))(2)}(2-)}] 14. The distortion of the rigid naphthalene and acenaphthene backbone away from ideal was investigated in each case and correlated in general with the steric bulk of the interacting atoms located at the proximal peri positions. Naturally, introduction of the ethane linker in acenaphthene compounds increased the splay of the bay region compared with equivalent naphthalene derivatives resulting in greater peri distances. The conformation of the aromatic rings and subsequent location of p-type lone pairs has a significant impact on the geometry of the peri region, with anomalies in peri separations correlated to the ability of the frontier orbitals to take part in attractive or repulsive interactions. In all but one of the monocations a quasi-linear three-body C(Me)-E···Z (E = Te, Se, S; Z = Br/E) fragment provides an attractive component for the E···Z interaction. Density functional studies confirmed these interactions and suggested the onset of formation of three-center, four-electron bonding under appropriate geometric conditions, becoming more prevalent as heavier congeners are introduced along the series. The increasingly large J values for Se-Se, Te-Se, and Te-Te coupling observed in the (77)Se and (125)Te NMR spectra for 1, 3, 4, 9, 10, and 13 give further evidence for the existence of a weakly attractive through-space interaction.

Transforming growth factor ß inhibits bone morphogenetic protein-induced transcription through novel phosphorylated Smad1/5-Smad3 complexes.

In vivo cells receive simultaneous signals from multiple extracellular ligands and must integrate and interpret them to respond appropriately. Here we investigate the interplay between pathways downstream of two transforming growth factor ß (TGF-ß) superfamily members, bone morphogenetic protein (BMP) and TGF-ß. We show that in multiple cell lines, TGF-ß potently inhibits BMP-induced transcription at the level of both BMP-responsive reporter genes and endogenous BMP target genes. This inhibitory effect requires the TGF-ß type I receptor ALK5 and is independent of new protein synthesis. Strikingly, we show that Smad3 is required for TGF-ß's inhibitory effects, whereas Smad2 is not. We go on to demonstrate that TGF-ß induces the formation of complexes comprising phosphorylated Smad1/5 and Smad3, which bind to BMP-responsive elements in vitro and in vivo and mediate TGF-ß-induced transcriptional repression. Furthermore, loss of Smad3 confers on TGF-ß the ability to induce transcription via BMP-responsive elements. Our results therefore suggest that not only is Smad3 important for mediating TGF-ß's inhibitory effects on BMP signaling but it also plays a critical role in restricting the transcriptional output in response to TGF-ß.

Exploring hypervalency and three-centre, four-electron bonding interactions: reactions of acenaphthene chalcogen donors and dihalogen acceptors.

Sterically crowded peri-substituted selenium and tellurium acenaphthene donors D1-D7 [Acenap(EPh)(Br) E = Se, Te; Acenap(SePh)(EPh) E = Se, S; Acenap(TePh)(EPh) E = S, Se, Te] react with dibromine and diiodine acceptors to afford a group of structurally diverse addition products 1-12, comparable in some cases to previously reported naphthalene analogues. Tellurium donors D4-D6 react conventionally with the dihalogens to afford insertion adducts 6-11 (X-R(2)Te-X) exhibiting molecular see-saw geometries, characterised by hypervalent X-Te-X quasi-linear fragments. The reactions of selenium donors D1-D3 with diiodine afford expected neutral charge-transfer (CT) spoke adducts 1, 4 and 5 (R(2)Se-I-I) containing quasi-linear Se-I-I alignments. Conversely, treatment of D2 and D3 with dibromine results in the formation of two tribromide salts 2 and 3 containing bromoselanyl cations [R(2)Se-Br](+)···[Br-Br(2)](-), each exhibiting a quasi-linear three-body Br-Se···E (E = Se, S) fragment. The peri-bonding in these species can be thought of as a weak hypervalent G···Se-X three-centre, four-electron (3c-4e) type interaction, closely related to the T-shaped 3c-4e interaction. Density-functional calculations performed on 2 and 3 and their bare cations (2a and 3a) reveal Wiberg bond indices of 0.25-0.37, suggesting substantial 3c-4e character in these systems. The presence of such an interaction operating in 2 and 3 alleviates steric strain within the peri-region and minimises the degree of molecular distortion required to achieve a relaxed geometry. Ditellurium donor D7 reacts with dibromine to afford an unorthodox insertion adduct 12 containing a Te-O-Te bridge and two quasi-linear Br-Te-O fragments, with the central tellurium atoms assuming a molecular see-saw geometry. Whilst DFT calculations indicate 12 is thermodynamically unfavourable, its formation is viable under experimental conditions.

Onset of three-centre, four-electron bonding in peri-substituted acenaphthenes: a structural and computational investigation.

Two series of sterically crowded peri-substituted acenaphthenes have been prepared, containing mixed halogen-chalcogen functionalities at the 5,6-positions in A1-A6 (Acenap[X][EPh] (Acenap = acenaphthene-5,6-diyl; X = Br, I; E = S, Se, Te) and chalcogen-chalcogen moieties in A7-A12 (Acenap[EPh][E'Ph] (Acenap = acenaphthene-5,6-diyl; E/E' = S, Se, Te). The related dihalide compounds A13-A16 Acenap[XX'] (XX' = BrBr, II, IBr, ClCl) have also been prepared. Distortion of the acenaphthene framework away from the ideal was studied as a function of the steric bulk of the interacting halogen and chalcogen atoms occupying the peri-positions. The acenaphthene series experiences a general increase in peri-separation for molecules accommodating heavier congeners and maps the trends observed previously for the analogous naphthalene compounds N1-N12 (Nap[X][EPh], Nap[EPh][E'Ph] (X = Br, I; E/E' = S, Se, Te). The conformation of the aromatic ring systems and subsequent location of p-type lone-pairs dominates the geometry of the peri-region. The differences in peri-separations observed for compounds adopting differing conformations of the peri-substituted phenyl group can be correlated to the ability of the frontier orbitals of the halogen or chalcogen atoms to take part in attractive or repulsive interactions. Density-functional studies have confirmed these interactions and suggested the onset of formation of three-centre, four-electron bonding under appropriate geometric conditions.

Recruitment of TIF1γ to chromatin via its PHD finger-bromodomain activates its ubiquitin ligase and transcriptional repressor activities.

The interplay between sequence-specific DNA-binding transcription factors, histone-modifying enzymes, and chromatin-remodeling enzymes underpins transcriptional regulation. Although it is known how single domains of chromatin "readers" bind specific histone modifications, how combinations of histone marks are recognized and decoded is poorly understood. Moreover, the role of histone binding in regulating the enzymatic activity of chromatin readers is not known. Here we focus on the TGF-ß superfamily transcriptional repressor TIF1γ/TRIM33/Ectodermin and demonstrate that its PHD finger-bromodomain constitutes a multivalent histone-binding module that specifically binds histone H3 tails unmethylated at K4 and R2 and acetylated at two key lysines. TIF1γ's ability to ubiquitinate its substrate Smad4 requires its PHD finger-bromodomain, as does its transcriptional repressor activity. Most importantly, TIF1γ's E3 ubiquitin ligase activity is induced by histone binding. We propose a model of TIF1γ activity in which it dictates the residence time of activated Smad complexes at promoters of TGF-ß superfamily target genes.

Mixed N-heterocyclic carbene/phosphite ruthenium complexes: towards a new generation of olefin metathesis catalysts.

The synthesis, characterisation and catalytic behaviour of ruthenium indenylidene complexes bearing an N-heterocyclic carbene and triisopropylphosphite are described.

An N-terminally truncated Smad2 protein can partially compensate for loss of full-length Smad2.

TGFbeta (transforming growth factor beta) superfamily signalling is critical both for early embryonic development and later for tissue homoeostasis in adult organisms. The use of gene-disruption techniques in mice has been essential to understanding the functional roles of the components of the pathways downstream of TGFbeta superfamily ligands, in particular, the receptors and the Smads that transduce signals from the plasma membrane to the nucleus. Smad2 functions downstream of TGFbeta, Activin and Nodal, and a number of Smad2 mutant mice have been generated by different laboratories. Although in all cases these Smad2-deficient mice were embryonic lethal, those created by deletion of the first coding exon survived longer than those generated by replacing part of the MH (Mad homology) 1 domain or deleting all or part of the MH2 domain. Moreover, they displayed a less severe phenotype, as they were capable of transiently inducing mesoderm. In the present study, we show that embryonic fibroblasts taken from the Smad2 mutant mice created by deletion of the first coding exon express a small amount of an N-terminally truncated Smad2 protein. We show this protein results from internal initiation at Met(241) and encodes the entire MH2 domain and the C-terminal part of the linker. We demonstrate that this protein is incorporated into Smad heteromeric complexes, can interact with DNA-binding transcription factors and thereby can mediate TGFbeta-induced transcriptional activation from a number of TGFbeta-responsive elements. We propose that this functional truncated Smad2 protein can partially compensate for the loss of full-length Smad2, thereby providing an explanation for the differing phenotypes of Smad2 mutant mice.

Transforming growth factor beta-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth.

Transforming growth factor beta (TGF-beta) signals predominantly through a receptor complex comprising ALK5 and TbetaRII to activate receptor-regulated Smads (R-Smads) Smad2 and Smad3. In endothelial cells, however, TGF-beta can additionally activate Smad1 and Smad5. Here, we report that TGF-beta also strongly induces phosphorylation of Smad1/5 in many different normal epithelial cells, epithelium-derived tumor cells, and fibroblasts. We demonstrate that TbetaRII and ALK5, as well as ALK2 and/or ALK3, are required for TGF-beta-induced Smad1/5 phosphorylation. We show that the simultaneous activation of the R-Smads Smad2/3 and Smad1/5 by TGF-beta results in the formation of mixed R-Smad complexes, containing, for example, phosphorylated Smad1 and Smad2. The prevalence of these mixed R-Smad complexes explains why TGF-beta-induced Smad1/5 phosphorylation does not result in transcriptional activation via bone morphogenetic protein (BMP)-responsive elements, which bind activated Smad1/5-Smad4 complexes that are induced by BMP stimulation. Thus, TGF-beta induces two parallel pathways: one signaling via Smad2-Smad4 or Smad3-Smad4 complexes and the other signaling via mixed R-Smad complexes. Finally, we assess the function of the novel arm of TGF-beta signaling and show that TGF-beta-induced Smad1/5 activation is not required for the growth-inhibitory effects of TGF-beta but is specifically required for TGF-beta-induced anchorage-independent growth.

Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif.

Transforming growth factor beta (TGF-beta) superfamily members signal via complexes of activated Smads, comprising phosphorylated receptor-regulated Smads, such as Smad2 and Smad3, and Smad4. These complexes are recruited to DNA by specific transcription factors. The forkhead/winged-helix transcription factors, XFast-1/XFoxH1a and XFast-3/XFoxH1b, bind an activated Smad heterotrimer comprising two Smad2s and one Smad4. Here we identify a novel Smad2 interaction motif, the Fast/FoxH1 motif (FM), present in all known Fast/FoxH1 family members, N-terminal to the common Smad interaction motif (SIM). The FM is necessary and sufficient to bind active Smad2/Smad4 complexes. The FM differs from the SIM since it discriminates between Smad2 and Smad3, and moreover only binds phosphorylated Smad2 in the context of activated Smad complexes. It is the first Smad interaction motif with this property. Site-directed mutagenesis indicates that the binding site for the FM on a Smad2/Smad4 heterotrimer is a hydrophobic pocket that incorporates the Smad/Smad interface. We demonstrate that the presence of an FM and SIM in the Fast/FoxH1 proteins allows them to compete efficiently for activated Smad2/Smad4 complexes with transcription factors such as Mixer that only contain a SIM. This establishes a hierarchy of Smad-interacting transcription factors, determined by their affinity for active Smad complexes.

Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif.

Transforming growth factor-beta (TGF-beta)/activin-induced Smad2/Smad4 complexes are recruited to different promoter elements by transcription factors, such as Fast-1 or the Mix family proteins Mixer and Milk, through a direct interaction between Smad2 and a common Smad interaction motif (SIM) in the transcription factors. Here we identify residues in the SIM critical for Mixer-Smad2 interaction and confirm their functional importance by demonstrating that only Xenopus and zebrafish Mix family members containing a SIM with all the correct critical residues can bind Smad2 and mediate TGF-beta-induced transcriptional activation in vivo. We identify significant sequence similarity between the SIM and the Smad-binding domain (SBD) of the membrane-associated protein SARA (Smad anchor for receptor activation). Molecular modelling, supported by mutational analyses of Smad2 and the SIM and the demonstration that the SARA SBD competes directly with the SIM for binding to Smad2, indicates that the SIM binds Smad2 in the same hydrophobic pocket as does the proline-rich rigid coil region of the SARA SBD. Thus, different Smad2 partners, whether cytoplasmic or nuclear, interact with the same binding pocket in Smad2 through a common proline-rich motif.