Significance of the Tks4 scaffold protein in bone tissue homeostasis

Abstract The main driver of osteoporosis is an imbalance between bone resorption and formation. The pathogenesis of osteoporosis has also been connected to genetic alterations in key osteogenic factors and dysfunction of bone marrow mesenchymal stem/stromal cells (BM-MSCs). Tks4 (encoded by the Sh3pxd2b gene) is a scaffold protein involved in podosome organization. Homozygous mutational inactivation of Sh3pxd2b causes Frank-ter Haar syndrome (FTHS), a genetic disease that affects bone tissue as well as eye, ear, and heart functions. To date, the role of Tks4 in adult bone homeostasis has not been investigated. Therefore, the aim of this study was to analyze the facial and femoral bone phenotypes of Sh3pxd2b knock-out (KO) mice using micro-CT methods. In addition to the analysis of the Sh3pxd2b-KO mice, the bone microstructure of an FTHS patient was also examined. Macro-examination of skulls from Tks4-deficient mice revealed craniofacial malformations that were very similar to symptoms of the FTHS patient. The femurs of the Sh3pxd2b-KO mice had alterations in the trabecular system and showed signs of osteoporosis, and, similarly, the FTHS patient also showed increased trabecular separation/porosity. The expression levels of the Runx2 and osteocalcin bone formation markers were reduced in the bone and bone marrow of the Sh3pxd2b-KO femurs, respectively. Our recent study demonstrated that Sh3pxd2b-KO BM-MSCs have a reduced ability to differentiate into osteoblast lineage cells; therefore, we concluded that the Tks4 scaffold protein is important for osteoblast formation, and that it likely plays a role in bone cell homeostasis

Structural insights into the tyrosine phosphorylation-mediated inhibition of SH3 domain-ligand interactions.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains have been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr-89 and Tyr-134 in ABL1, or the homologous residues Tyr-116 and Tyr-161 in ABL2 induce only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine-phosphorylation induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed that the residues phosphorylated in our model systems are not only well conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome

The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages

The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation

Az ENVASSO Projekt által kidolgozott módszertan tesztelése az erózió okozta talaj biodiverzitás csökkenés mérésére

Az Európai Unió 2002-es „Egy tematikus talajvédelmi stratégia felé” című közleményében definiálta a talaj funkcióit és a talajainkat veszélyeztető legfontosabb nyolc tényezőt, melyek a következők: erózió, talaj szervesanyag-csökkenés, talajszennyezés, talajlefedés, talajtömörödés, talaj biodiverzitás csökkenés, szikesedés, árvizek és földcsuszamlások. Az „Environmental Assessment of Soil for Monitoring” (ENVASSO) Projekt az EU 25 tagállamának részvételével, egy harmonizált módszertan kidolgozását tűzte ki célul a talajtulajdonságokban bekövetkező változások nyomon követésére. Ennek érdekében minden egyes talajt veszélyeztető tényező vizsgálatára egy indikátor hármast (TOP3) dolgozott ki. A talajainkat veszélyeztető nyolc tényező közül a talaj biodiverzitás csökkenésével foglalkozunk részletesen, melynek legfontosabb kiválasztott indikátorai: a földigiliszta (BIO1) és ugróvillás (BIO2) diverzitás (fajdiverzitás), és a mikrobiológiai talajlégzés (BIO3) (biológiai funkció). Kutatásunk célkitűzése az volt, hogy az ENVASSO által javasolt módszertan kivitelezhetőségét teszteljük a Szent István Egyetem Tangazdaságában egy eróziós katéna mentén. Megállapítottuk, hogy a talaj biodiverzitása csökkent az erózió mértékének növekedésével, valamint a módszertan megfelelő tervezéssel alkalmas monitoring célra. | The communiqué released by the European Commission in 2002, entitled “Towards a Thematic Strategy on Soil Protection”, defined soil functions and the eight most important threats to soil. These were: soil erosion, the decline of soil organic matter, soil contamination, soil sealing, soil compaction, the decline of soil biodiversity, soil salinisation, and floods and landslides. With the participation of 25 EU member countries, the “Environmental Assessment of Soil for Monitoring” (ENVASSO) Project was set up to elaborate a harmonized methodology for monitoring changes in soil properties. For this purpose the three most important indicators (TOP3) were designated for each factor threatening the soil. This paper is concerned chiefly with the decline in soil biodiversity, where the indicators selected were: the species diversity of earthworms (BIO1) and springtails (BIO2) and the microbiological soil respiration (BIO3). The ENVASSO guidelines recommend that these indicators should be monitored preferably every three years, but certainly at least every five years. If possible, samples should always be taken in the same season (spring or autumn) to facilitate the monitoring of changes over time. The ENVASSO methodology gives a clear description of sampling tools and methods, chemicals, and laboratory methods. The ISO standards, used alongside the ENVASSO methodology, also provide useful guidelines for sampling and laboratory analysis. The aim of the work was to test the methodology recommended by the ENVASSO Project to determine the ease of application and the time and labour requirements for monitoring the population and species composition of earthworms and springtails and for recording microbial soil respiration. A testing area was designated at the experimental farm of Szent István University in Józsefmajor, where four soil profiles were analysed, located along an erosion gradient: 1. erosion-free; 2. slightly eroded; 3. greatly eroded; 4. sedimentation area. Field samples for the analysis of the population and species composition of earthworms and springtails and for recording microbial soil respiration were taken in May 2007 on all four soil profiles, as illustrated in Figure 1. Laboratory analysis was carried out according to the ISO standards and the ENVASSO methodology. All in all, it could be concluded that the indicators chosen in the ENVASSO Project and used in the present work (BIO1, BIO2, BIO3) gave a satisfactory picture of the decline in soil biodiversity and can thus be regarded as parameters providing a clear illustration of the deterioration in soil status

EGF Regulates the Interaction of Tks4 with Src through Its SH2 and SH3 Domains

The nonreceptor tyrosine kinase Src is a central component of the epidermal growth factor (EGF) signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues 466–474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues 508–511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to a synergistic enhancement of binding strength and a highly stable, “bidentate”-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least 3 h in living cells. We conclude that the interaction of Tks4 with Src may result in the long term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation