Molecular characterization and expression of TGFßRI and TGFßRII and its association with litter size in Tibetan sheep.

BACKGROUNDS: Transforming growth factor-ß (TGF-ß) type I receptor (TGFßRI) and type II receptor (TGFßRII) are the members of the TGFß superfamily, which are potent regulators of cell proliferation and differentiation in many organ systems, and they play key roles in multiple aspects of follicle development. OBJECTIVES: We aimed to explore the characterization, expression analysis of TGFßRI and TGFßRII genes, and the association with litter size in Tibetan sheep. METHODS: In this study, we cloned the complete coding sequences of TGFßRI and TGFßRII genes in Tibetan sheep and analyzed their genomic structures. RESULTS: The results showed that percentages of sequences homology of the two proteins in Tibetan sheep were the most similar to Ovis aries (100%), followed by Bos mutus (99%). The RT-qPCR showed that two genes were expressed widely in the different tissues of Tibetan sheep. The TGFßRI expression was the highest in the lung (p < 0.05), followed by the spleen and ovary (p < 0.05). The TGFßRII expression was significantly higher in uterus than that in lung and ovary (p < 0.05). In addition, the χ2 test indicated that all ewes in the population were in Hardy-Weinberg equilibrium, and the population was in medium or low polymorphic information content status. We also found four Single Nucleotide Polymorphism (SNPs), g.9414A > G, g.28881A > G, g.28809T > C, g.10429G > A in sheep TGFßRI gene and g.63940C > T, g.63976C > T, g.64538C > T, g.64504T > A in TGFßRII gene. Three genotypes, except for g.64504T > A, and three haplotypes were identified in each gene. linkage disequilibrium analysis indicated that there was strong linkage disequilibrium in each gene. The association analysis showed that the four SNPs of TGFßRI were associated with litter size (p < 0.05), and g.63940C > T of TGFßRII was confirmed to be associated with litter size (p < 0.05). CONCLUSIONS: Based on these preliminary results, we can assume that TGFß receptors (TGFßRI and TGFßRII) may play an important role in sheep reproduction.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Evaluating Deep Learning models for predicting ALK-5 inhibition.

Computational methods have been widely used in drug design. The recent developments in machine learning techniques and the ever-growing chemical and biological databases are fertile ground for discoveries in this area. In this study, we evaluated the performance of Deep Learning models in comparison to Random Forest, and Support Vector Regression for predicting the biological activity (pIC50) of ALK-5 inhibitors as candidates to treat cancer. The generalization power of the models was assessed by internal and external validation procedures. A deep neural network model obtained the best performance in this comparative study, achieving a coefficient of determination of 0.658 on the external validation set with mean square error and mean absolute error of 0.373 and 0.450, respectively. Additionally, the relevance of the chemical descriptors for the prediction of biological activity was estimated using Permutation Importance. We can conclude that the forecast model obtained by the deep neural network is suitable for the problem and can be employed to predict the biological activity of new ALK-5 inhibitors.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5.

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases, and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases, and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homologue 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

Structure-Based Virtual Screening, Molecular Dynamics and Binding Free Energy Calculations of Hit Candidates as ALK-5 Inhibitors.

Activin-like kinase 5 (ALK-5) is involved in the physiopathology of several conditions, such as pancreatic carcinoma, cervical cancer and liver hepatoma. Cellular events that are landmarks of tumorigenesis, such as loss of cell polarity and acquisition of motile properties and mesenchymal phenotype, are associated to deregulated ALK-5 signaling. ALK-5 inhibitors, such as SB505154, GW6604, SD208, and LY2157299, have recently been reported to inhibit ALK-5 autophosphorylation and induce the transcription of matrix genes. Due to their ability to impair cell migration, invasion and metastasis, ALK-5 inhibitors have been explored as worthwhile hits as anticancer agents. This work reports the development of a structure-based virtual screening (SBVS) protocol aimed to prospect promising hits for further studies as novel ALK-5 inhibitors. From a lead-like subset of purchasable compounds, five molecules were identified as putative ALK-5 inhibitors. In addition, molecular dynamics and binding free energy calculations combined with pharmacokinetics and toxicity profiling demonstrated the suitability of these compounds to be further investigated as novel ALK-5 inhibitors.

Identification of a transforming growth factor-ß type I receptor transcript in Eriocheir sinensis and its molting-related expression in muscle tissues.

The transforming growth factor-ß (TGF-ß) signaling pathway is conserved across animals, and knowledge of its roles during the molt cycle in crustaceans is presently very limited. This study investigates the roles of the TGF-ß receptor in molting-related muscle growth in Eriocheir sinensis. Using the RT-PCR and RACE techniques, we obtained a 1722 bp cDNA sequence encoding a transforming growth factor-ß type I receptor in Eriocheir sinensis, designated EsTGFBRI, which contains a 124 bp 5'-untranslated region, a 20 bp partial 3'-untranslated region and a 1578 bp open reading frame encoding 525 amino acids. The deduced EsTGFBRI contains an N-terminal 24 amino acid signal peptide, an activin type I and II receptor domain, a transmembrane helix region, a glycine-serine-rich motif, and a conserved serine/threonine kinase catalytic domain including an activation loop. The qRT-PCR results showed that EsTGFBRI gene was highly expressed in the intermolt testis and ovary in mature crabs. In juvenile crabs, the mRNA levels of EsTGFBRI in claw and abdominal muscles in the later premolt D3-4 stage were significantly higher than those in the intermolt C and postmolt A-B stages. There was no significant change in EsTGFBRI mRNA levels in walking leg muscles during the molt cycle. The results suggest that EsTGFBRI is probably play roles in molting-related muscle growth in E. sinensis. This study provides a necessary basis for elucidating the functions of TGF-ß-like signaling mediated by TGFBRI in molting-related muscle growth in crustaceans.

PTPN3 acts as a tumor suppressor and boosts TGF-ß signaling independent of its phosphatase activity.

TGF-ß controls a variety of cellular functions during development. Abnormal TGF-ß responses are commonly found in human diseases such as cancer, suggesting that TGF-ß signaling must be tightly regulated. Here, we report that protein tyrosine phosphatase non-receptor 3 (PTPN3) profoundly potentiates TGF-ß signaling independent of its phosphatase activity. PTPN3 stabilizes TGF-ß type I receptor (TßRI) through attenuating the interaction between Smurf2 and TßRI. Consequently, PTPN3 facilitates TGF-ß-induced R-Smad phosphorylation, transcriptional responses, and subsequent physiological responses. Importantly, the leucine-to-arginine substitution at amino acid residue 232 (L232R) of PTPN3, a frequent mutation found in intrahepatic cholangiocarcinoma (ICC), disables its role in enhancing TGF-ß signaling and abolishes its tumor-suppressive function. Our findings have revealed a vital role of PTPN3 in regulating TGF-ß signaling during normal physiology and pathogenesis.

ALK4/5-dependent TGF-ß signaling contributes to the crosstalk between neurons and microglia following axonal lesion.

Neuronal activity is closely influenced by glia, especially microglia which are the resident immune cells in the central nervous system (CNS). Microglia in medicinal leech are the only cells able to migrate to the injury site within the 24 hours post-lesion. The microglia-neuron interactions constitute an important mechanism as there is neither astrocyte nor oligodendrocyte in the leech CNS. Given that axonal sprouting is impaired when microglia recruitment is inhibited, the crosstalk between microglia and neurons plays a crucial role in neuroprotection. The present results show that neurons and microglia both use ALK4/5 (a type of TGF-ß receptor) signaling in order to maintain mutual exchanges in an adult brain following an axonal injury. Indeed, a TGF-ß family member (nGDF) is immediately released by injured axons contributing to the early recruitment of ALK4/5+ microglia to the lesion site. Surprisingly, within the following hours, nGDF from microglia activates ALK4/5+ neurons to maintain a later microglia accumulation in lesion. Taken together, the results demonstrate that ALK4/5 signaling is essential throughout the response to the lesion in the leech CNS and gives a new insight in the understanding of this pathway. This latter is an important signal contributing to a correct sequential mobilization over time of microglia recruitment leading to axon regeneration.

Dihydromyricetin attenuates hypertrophic scar formation by targeting activin receptor-like kinase 5.

Hypertrophic scar (HPS) is a manifestation of abnormal tissue repair, representing excessive extracellular matrix production and abnormal function of fibroblasts, for which no satisfactory treatment is available at present. Here we identified a natural product of flavonoid, dihydromyricetin, could effectively attenuate HPS formation. We showed that local intradermal injection of dihydromyricetin (50 µM) reduced the gross scar area, cross-sectional size of the scar and the scar elevation index in a mechanical load-induced mouse model. In addition, dihydromyricetin treatment also markedly decreased collagen density of the scar tissue. Furthermore, both in vitro and in vivo study both demonstrated that dihydromyricetin inhibited the proliferation, activation, contractile and migration abilities of hypertrophic scar-derived fibroblasts (HSFs) but did not affect HSFs apoptosis. Western blot analysis revealed that dihydromyricetin could down-regulate the phosphorylation of Smad2 and Smad3 of TGF-ß signaling. Such bioactivity of dihydromyricetin may result from its selective binding to the catalytic region of activin receptor-like kinase 5 (ALK5), as suggested by the molecular docking study and kinase binding assay (12.26 µM). Above all, dihydromyricetin may prove to be a promising agent for the treatment of HPS and other fibroproliferative disorders.

Novel Transforming Growth Factor-Beta Receptor 1 Antagonists through a Pharmacophore-Based Virtual Screening Approach.

As new drugs for the treatment of malignant tumors, transforming growth factor-beta receptor 1 (TGFßR1) antagonists have attracted wide attention. Based on the crystal structure of TGFßR1-BMS22 complex, the pharmacophore model A02 with two hydrogen bond acceptors (HBAs) and four hydrophobic (HYD) properties was constructed. From the common features of active ligands reported in the literature, pharmacophore model B10 was also generated, which has two aromatic ring centers (RAs) and two HYD properties. The two models have high sensitivity and specificity to the training set, and they are highly consistent in spatial structure. Combining the two pharmacophore models, two novel skeleton structures with potential activity were selected by virtual screening from the DruglikeDiverse, MiniMaybridge, and ZINC Drug-Like databases. Four compounds (YXY01⁻YXY04) with potential anti-TGFßR1 activity were designed based on the new skeleton structures. In combination with Lipinski's rules; absorption, distribution, metabolism, excretion, and toxicity (ADMET); and, toxicological properties predicted in the study, YXY01-03 with the novel skeleton, good drug-like properties, and potential activity were finally discovered and may have higher safety relative to BMS22, which may be valuable for further research.

A TGF-ß type I receptor-like molecule with a key functional role in Haemonchus contortus development.

Here we investigated the gene of a transforming growth factor (TGF)-ß type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-ß family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-ß type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-ß type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.

Structure, molecular dynamics simulation, and docking studies of Dictyostelium discoideum and human STRAPs.

The Serine Threonine kinase Receptor Associated Protein (STRAP) is a WD40 containing protein that provides a platform for protein interactions during cell proliferation and development. Overexpression and misregulation of STRAP contributes to various carcinomas that are now recognized as therapeutic targets especially for colorectal and lung cancers. The present study was undertaken to find an effective drug against this molecule using a simple system like Dictyostelium discoideum; which shares close homology to humans. Using techniques like structural modeling, molecular dynamics (MD) simulation and molecular docking, we found similar structure and dynamic behaviors in both, except for the presence of dissimilar numbers of ß-sheets and loop segments. We identified a novel and potential drug targeted to STRAP. The results obtained allow us to use Dictyostelium as a model system for further in vivo studies. Finally, the results of protein-protein interactions using molecular docking and essential dynamics studies show STRAP to participate in TGF-ß signaling in humans. Further, we show some structural units that govern the interaction of TGFß-RI with STRAP and Smad7 proteins in TGF-ß signaling pathway. In conclusion, we propose that D. discoideum can be used for enhancing our knowledge about STRAP protein.

2D-QSAR study, molecular docking, and molecular dynamics simulation studies of interaction mechanism between inhibitors and transforming growth factor-beta receptor I (ALK5).

Transforming growth factor type 1 receptor (ALK5) is kinase associated with a wide variety of pathological processes, and inhibition of ALK5 is a good strategy to treat many kinds of cancer and fibrotic diseases. Recently, a series of compounds have been synthesized as ALK5 inhibitors. However, the study of their selectivity against other potential targets remains elusive. In this research, a data-set of ALK5 inhibitors were collected and studied based on the combination of 2D-QSAR, molecular docking and molecular dynamics simulation. The quality of QSAR models were assessed statistically by F, R2, and R2ADJ, proved to be credible. The cross-validations for the models (q2LOO = 0.571 and 0.629, respectively) showed their robustness, while the external validations (r2test = 0.703 and 0.764, respectively) showed their predictive power. Besides, the predicted binding free energy results calculated by MM/GBSA method were in accordance with the experimental data, and the van der Waals energy term was the factor that had the most significant impact on ligand binding. What is more, several important residues were found to significantly affect the binding affinity. Finally, based on our analyses above, a proposed series of molecules were designed.

Computational analyses of interactions between ALK-5 and bioactive ligands: insights for the design of potential anticancer agents.

Activin Receptor-Like Kinase 5 (ALK-5) is related to some types of cancer, such as breast, lung, and pancreas. In this study, we have used molecular docking, molecular dynamics simulations, and free energy calculations in order to explore key interactions between ALK-5 and six bioactive ligands with different ranges of biological activity. The motivation of this work is the lack of crystal structure for inhibitor-protein complexes for this set of ligands. The understanding of the molecular structure and the protein-ligand interaction could give support for the development of new drugs against cancer. The results show that the calculated binding free energy using MM-GBSA, MM-PBSA, and SIE is correlated with experimental data with r2 = 0.88, 0.80, and 0.94, respectively, which indicates that the calculated binding free energy is in excellent agreement with experimental data. In addition, the results demonstrate that H bonds with Lys232, Glu245, Tyr249, His283, Asp351, and one structural water molecule play an important role for the inhibition of ALK-5. Overall, we discussed the main interactions between ALK-5 and six inhibitors that may be used as starting points for designing new molecules to the treatment of cancer.