Finale: the last minutes of Smads.

TGF-beta ligands induce phosphorylation of receptor-activated Smads at both the C-terminal tail and the linker region. Two papers from Massagué and colleagues (Alarcón et al., 2009; Gao et al., 2009) reveal a dual role for this linker phosphorylation, which is required for activation of Smads and for their degradation.

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways.

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways.

Membrane targeting of inhibitory Smads through palmitoylation controls TGF-ß/BMP signaling.

TGF-ß/BMP (bone morphogenetic protein) signaling pathways play conserved roles in controlling embryonic development, tissue homeostasis, and stem cell regulation. Inhibitory Smads (I-Smads) have been shown to negatively regulate TGF-ß/BMP signaling by primarily targeting the type I receptors for ubiquitination and turnover. However, little is known about how I-Smads access the membrane to execute their functions. Here we show that Dad, the Drosophila I-Smad, associates with the cellular membrane via palmitoylation, thereby targeting the BMP type I receptor for ubiquitination. By performing systematic biochemistry assays, we characterized the specific cysteine (Cys556) essential for Dad palmitoylation and membrane association. Moreover, we demonstrate that dHIP14, a Drosophila palmitoyl acyl-transferase, catalyzes Dad palmitoylation, thereby inhibiting efficient BMP signaling. Thus, our findings uncover a modification of the inhibitory Smads that controls TGF-ß/BMP signaling activity.

Structural determinants of Smad function in TGF-ß signaling.

Smad transcription factors are central to the signal transduction pathway that mediates the numerous effects of the transforming growth factor ß (TGF-ß) superfamily of cytokines in metazoan embryo development as well as in adult tissue regeneration and homeostasis. Although Smad proteins are conserved, recent genome-sequencing projects have revealed their sequence variation in metazoan evolution, human polymorphisms, and cancer. Structural studies of Smads bound to partner proteins and target DNA provide a framework for understanding the significance of these evolutionary and pathologic sequence variations. We synthesize the extant mutational and structural data to suggest how genetic variation in Smads may affect the structure, regulation, and function of these proteins. We also present a web application that compares Smad sequences and displays Smad protein structures and their disease-associated variants.

TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility.

Ligands of the transforming growth factor-beta (TGFbeta) superfamily of growth factors initiate signal transduction through a bewildering complexity of ligand-receptor interactions. Signalling then converges to nuclear accumulation of transcriptionally active SMAD complexes and gives rise to a plethora of specific functional responses in both embryos and adult organisms. Current research is focused on the mechanisms that regulate SMAD activity to evoke cell-type-specific and context-dependent transcriptional programmes. An equally important challenge is understanding the functional role of signal strength and duration. How are these quantitative aspects of the extracellular signal regulated? How are they then sensed and interpreted, and how do they affect responses?

A Smad action turnover switch operated by WW domain readers of a phosphoserine code.

When directed to the nucleus by TGF-ß or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-ß pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-ß signal delivery to turnover of the Smad signal transducers.

Antifibrotic Effect of Smad Decoy Oligodeoxynucleotide in a CCl4-Induced Hepatic Fibrosis Animal Model.

Hepatic fibrosis is the wound-healing process of chronic hepatic disease that leads to the end-stage of hepatocellular carcinoma and demolition of hepatic structures. Epithelial⁻mesenchymal transition (EMT) has been identified to phenotypic conversion of the epithelium to mesenchymal phenotype that occurred during fibrosis. Smad decoy oligodeoxynucleotide (ODN) is a synthetic DNA fragment containing a complementary sequence of Smad transcription factor. Thus, this study evaluated the antifibrotic effects of Smad decoy ODN on carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. As shown in histological results, CCl4 treatment triggered hepatic fibrosis and increased Smad expression. On the contrary, Smad decoy ODN administration suppressed fibrogenesis and EMT process. The expression of Smad signaling and EMT-associated protein was markedly decreased in Smad decoy ODN-treated mice compared with CCl4-injured mice. In conclusion, these data indicate the practicability of Smad decoy ODN administration for preventing hepatic fibrosis and EMT processes.

Molecular characterization of two distinct Smads gene and their roles in the response to bacteria change and wound healing from Hyriopsis cumingii.

The proteins of Smad family are critical components of the TGF-ß superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF-ß signaling, Smad3 and Smad5, from the pearl mussel Hyriopsis cumingii. The full length cDNA of HcSmad3 and HcSmad5 were 2052 bp and 1908 bp and encoded two polypeptides of 418 and 461amino acid residues, respectively. The deduced amino acid of HcSmad3 and HcSmad5 possessed two putative conserved domains, MH1 and MH2, a conserved phosphorylation motif SSXS at the carboxyl-terminal. The two Smad genes were detected muscle, mantle, hepatopancreas and gill, but with a very low level in heamocytes. The transcripts of Smad3 and Smad5 were up-regulated in hemocytes and hepatopancreas after A. hydrophila and PGN stimulation. However, the expression of Smad3 and Smad5 were only up-regulated in hepatopancreas after A. hydrophila stimulation. The transcripts of Smad3 and Smad5 had a slight change in hepatopancreas after PGN stimulation. The transcripts of HcSmad3 showed very little increase and HcSmad5 mRNA significantly up-regulated after wounding.

Structure of the N-terminal domain of the protein Expansion: an 'Expansion' to the Smad MH2 fold.

Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-ß signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Šresolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-ß signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

Rational design of YAP WW1 domain-binding peptides to target TGFß/BMP/Smad-YAP interaction in heterotopic ossification.

The transforming growth factor-ß/bone morphogenic protein/Smad signaling pathway has been raised as a new and promising therapeutic target of heterotopic ossification, which is mediated by recruitment of transcription coactivator Yes-associated protein (YAP) to Smad. Here, we described a successful integration of computational modeling and experimental assay to rationally design novel peptide aptamers to disrupt YAP-Smad interaction by targeting YAP WW1 domain. In the protocol, a computational genetic evolution strategy was used to improve a population of potential YAP WW1-binding peptides generated from the YAP-recognition site in Smad protein, from which several promising peptides were selected and their affinities toward YAP WW1 domain were determined using binding assay. In addition, a high-activity peptide was further optimized based on its complex structure with YAP WW1 domain to derive a number of derivative peptides with higher binding potency to the domain. We also found that a strong YAP WW1 binder should have a negatively charged N-terminus, a positively charged C-terminus and a nonpolar core to match the electrostatic distribution pattern in peptide-binding pocket of YAP WW1 domain, which may also form additional nonbonded interactions such as hydrogen bond, salt bridge and π-π stacking to confer stability and specificity for the domain-peptide recognition.

A novel SMAD family protein, SMAD9 is involved in follicular initiation and changes egg yield of geese via synonymous mutations in exon1 and intron2.

Elevation of egg performance is vital to goose farming. Many poultry scientists are seeking for efficient molecular genetic markers associated with egg yield. In this study, mRNA differential display was adopted to investigate gene expression profiling in the follicular development of goose. For the first time, a novel SMAD family protein SMAD9 (EST CJ111007) was found to be involved in follicular initiation and used to be a candidate gene. Functional regions analysis of Smad9 indicated that SMAD9 protein is highly conserved in MH1 and MH2 domains, and the connection area is highly variable region. 6 pairs of primers (p1-p6) were designed to detect the SNPs of Smad9 by PCR-SSCP method. The results revealed that polymorphisms were discovered in the PCR products amplified with P1 primers in exon1 and P3 primers in intron2. In Smad9 exon1, 5 genotypes were found: FK, FF, JJ, JK and KK, including 2 SNPs: 243 bp G → A, 309 bp T → G, the mutations did not result in amino acid mutations; In intron2, 3 genotypes were found: AA, BB and AB, only 1 SNP (C → T). The annual egg yield of FK genotype geese or allele gene A in intron2 are significantly more than those of other genotypes on the average (p < 0.05). Taken together, it is suggested that Smad9 is a promising candidate gene affecting egg performance in goose.

Myotubularin-related protein 4 (MTMR4) attenuates BMP/Dpp signaling by dephosphorylation of Smad proteins.

Bone morphogenetic proteins (BMPs) signaling essentially regulates a wide range of biological responses. Although multiple regulators at different layers of the receptor-effectors axis have been identified, the mechanisms of homeostatic BMP signaling remain vague. Herein we demonstrated that myotubularin-related protein 4 (MTMR4), a FYVE domain-containing dual-specificity protein phosphatase (DUSP), preferentially associated with and dephosphorylated the activated R-Smads in cytoplasm, which is a critical checkpoint in BMP signal transduction. Therefore, transcriptional activation by BMPs was tightly controlled by the expression level and the intrinsic phosphatase activity of MTMR4. More profoundly, ectopic expression of MTMR4 or its Drosophila homolog CG3632 genetically interacted with BMP/Dpp signaling axis in regulation of the vein development of Drosophila wings. By doing so, MTMR4 could interact with and dephosphorylate Mothers against Decapentaplegic (Mad), the sole R-Smad in Drosophila BMP pathway, and hence affected the target genes expression of Mad. In conclusion, this study has suggested that MTMR4 is a necessary negative modulator for the homeostasis of BMP/Dpp signaling.

Smad inhibition by the Ste20 kinase Misshapen.

The level of TGF-ß/bone morphogenetic protein (BMP) signaling through Smad is tightly regulated to ensure proper embryonic patterning and homeostasis. Here we show that Smad activation by TGF-ß/BMP is blocked by a highly conserved phosphorylation event in the α-helix 1 region of Smad [T312 in Drosophila Smad1 (MAD)]. α-helix 1 phosphorylation reduces Smad interaction with TGF-ß/BMP receptor kinase and affects all receptor-activated Smads except Smad3. Tissue culture and transgenic studies in Drosophila further demonstrate that the biological activity of MAD is repressed by T312 phosphorylation in vivo. Through RNAi screening of the kinome, we have identified Misshapen (Msn) and the mammalian orthologs TNIK, MINK1, and MAP4K4 as the kinases responsible for α-helix 1 phosphorylation. Targeted expression of an active form of Msn in the wing imaginal disk disrupted activation of endogenous MAD by Dpp and expression of the Dpp/MAD target gene. Msn kinases belong to the Ste20 kinase family that has been shown to act as MAP kinase kinase kinase kinase (MAP4K). Our findings thus reveal a function of Msn independent of its impact on MAP kinase cascades. This Smad inhibition mechanism by Msn likely has important implications for development and disease.

Smad linker region phosphorylation in the regulation of extracellular matrix synthesis.

The canonical TGF-ß signalling pathway involves Smad transcription factors through direct serine phosphorylation of the carboxy termini, nuclear translocation and regulation of transcription by receptor-regulated (R)-Smad complexes. Smads can also be phosphorylated in the linker region most prominently by the action of mitogen-activated protein (MAP) kinases, which in turn have been activated by TGF-ß or a multitude of other growth factors and hormones. Linker region phosphorylation can prevent nuclear translocation of Smads and inhibit TGF-ß signalling, potentially leading to oncogenesis. However, some evidence has revealed that linker region phosphorylated Smads can be translocated to the nucleus where they regulate transcription particularly of the synthesis of extracellular matrix molecules. Matrix molecules such as collagen and proteoglycans are involved in diseases such a fibrosis and atherosclerosis, respectively, and the involvement of linker region phosphorylation may represent a new therapeutic target.

Multi-Harmony: detecting functional specificity from sequence alignment.

Many protein families contain sub-families with functional specialization, such as binding different ligands or being involved in different protein-protein interactions. A small number of amino acids generally determine functional specificity. The identification of these residues can aid the understanding of protein function and help finding targets for experimental analysis. Here, we present multi-Harmony, an interactive web sever for detecting sub-type-specific sites in proteins starting from a multiple sequence alignment. Combining our Sequence Harmony (SH) and multi-Relief (mR) methods in one web server allows simultaneous analysis and comparison of specificity residues; furthermore, both methods have been significantly improved and extended. SH has been extended to cope with more than two sub-groups. mR has been changed from a sampling implementation to a deterministic one, making it more consistent and user friendly. For both methods Z-scores are reported. The multi-Harmony web server produces a dynamic output page, which includes interactive connections to the Jalview and Jmol applets, thereby allowing interactive analysis of the results. Multi-Harmony is available at http://www.ibi.vu.nl/ programs/shmrwww.

Transforming growth factor ß1/Smad signalling pathway of aortic disorders: histopathological and immunohistochemical studies.

BACKGROUND: The aim of the present study was to evaluate the expressions and biological functions of the TGF-ß(1)/Smad signalling pathway of aortic disorders by way of histopathological and immunohistochemical studies. MATERIAL AND METHODS: Aortic specimens of 20 patients with aortic dissection, 9 patients with aortic aneurysm, 9 patients with coronary artery disease, and 5 deceased healthy adults were collected. The samples were stained with haematoxylin -eosin, Masson's trichrome, van Gieson, and alcian blue, and with immunohistochemical stainings to detect TGF-ß(1), type I receptor (TßRI), Smad2/3, Smad4, and Smad7. RESULTS: Masson's trichrome and van Gieson stainings showed attenuated collagens in the aorta of the patients with aortic dissection and aortic aneurysm. TGF-ß(1), TßRI, and Smad2/3 mainly showed a cytoplasmic immunoreactivity in the aortic media, Smad4 immunoreactivity was predominantly located in the cytoplasm and/or the nucleus of the aortic media, and Smad7 immunoreactivity was present in the nucleus of the aortic media and intima. The TGF-ß(1) signalling pathway proteins were similarly expressed in the aorta of aortic dissection and aortic aneurysm patients, while they were less pronounced in the aorta of coronary artery disease patients, and weak or negative in the aorta of healthy control individuals. CONCLUSIONS: These observations support the notion that there is an association between the TGF-ß(1)/Smad pathway and the pathological events of the aorta. Dysregulation of the TGF-ß(1)/Smad pathway may predispose the pathologenesis of aortic disorders.

A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

Identification, Molecular Characterization, and Tissue Expression Profiles of Three Smad Genes from Water Buffalo (Bubalus bubalis).

Smads are involved in a variety of biological activities by mediating bone morphogenetic protein (BMP) signals. The full-length coding sequences (CDSs) of buffalo Smads 1, 4, and 5 were isolated and identified through RT-PCR in this study. Their lengths are 1398 bp, 1662 bp, and 1398 bp, respectively. In silico analysis showed that their transcriptional region structures, as well as their amino acid sequences, physicochemical characteristics, motifs, conserved domains, and three-dimensional structures of their encoded proteins are highly consistent with their counterparts in the species of Bovidae. The three Smad proteins are all hydrophilic without the signal peptides and transmembrane regions. Each of them has an MH1 domain and an MH2 domain. A nuclear localization sequence was found in the MH1 domain of buffalo Smads 1 and 5. Prediction showed that the function of the three Smads is mainly protein binding, and they can interact with BMPs and their receptors. The three genes were expressed in all 10 buffalo tissues assayed, and their expression in the mammary gland, gonad, and spleen was relatively high. The results here indicate that the three buffalo Smads may be involved in the transcriptional regulation of genes in a variety of tissues.

Discovery and biological evaluation of phthalazines as novel non-kinase TGFß pathway inhibitors.

TGFß is crucial for the homeostasis of epithelial and neural tissues, wound repair, and regulating immune responses. Its dysregulation is associated with a vast number of diseases, of which modifying the tumor microenvironment is one of vital clinical interest. Despite various attempts, there is still no FDA-approved therapy to inhibit the TGFß pathway. Major mainstream approaches involve impairment of the TGFß pathway via inhibition of the TGFßRI kinase. With the purpose to identify non-receptor kinase-based inhibitors to impair TGFß signaling, an in-house chemical library was enriched, through a computational study, to eliminate TGFßRI kinase activity. Selected compounds were screened against a cell line engineered with a firefly luciferase gene under TGFß-Smad-dependent transcriptional control. Results indicated moderate potency for a molecule with phthalazine core against TGFß-Smad signaling. A series of phthalazine compounds were synthesized and evaluated for potency. The most promising compound (10p) exhibited an IC50 of 0.11 ± 0.02 µM and was confirmed to be non-cytotoxic up to 12 µM, with a selectivity index of approximately 112-fold. Simultaneously, 10p was confirmed to reduce the Smad phosphorylation using Western blot without exhibiting inhibition on the TGFßRI enzyme. This study identified a novel small-molecule scaffold that targets the TGFß pathway via a non-receptor-kinase mechanism.