Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

NMR Relaxation Dispersion Experiments to Study Phosphopeptide Recognition by SH2 Domains: The Grb2-SH2-Phosphopeptide Encounter Complex.

Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of 15N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the protein at a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient "touching" of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2-pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.

Csk αC Helix: A Computational Analysis of an Essential Region for Conformational Transitions.

Conformational changes are an essential feature for the function of some dynamic proteins. Understanding the mechanism of such motions may allow us to identify important properties, which may be directly related to the regulatory function of a protein. Also, this knowledge may be employed for a rational design of drugs that can shift the balance between active and inactive conformations, as well as affect the kinetics of the activation process. Here, the conformational changes in carboxyl-terminal Src kinase, the major catalytic repressor to the Src family of kinases, was investigated, and it was proposed as a functionally related hypothesis. A Cα Structure-Based Model (Cα-SBM) was applied to provide a description of the overall conformational landscape and further analysis complemented by detailed molecular dynamics simulations. As a first approach to Cα-SBM simulations, reversible transitions between active (closed) and inactive (open) forms were modeled as fluctuations between these two energetic basins. It was found that, in addition to the interdomain Carboxyl-terminal SRC Kinase (Csk) correlated motions, a conformational change in the αC helix is required for a complete conformational transition. The result reveals this as an important region of transition control and domain coordination. Restrictions in the αC helix region of the Csk protein were performed, and the analyses showed a direct correlation with the global conformational changes, with this location being propitious for future studies of ligands. Also, the Src Homology 3 (SH3) and SH3 plus Src Homology 2 (SH2) domains were excluded for a direct comparison with experimental results previously published. Simulations where the SH3 was deleted presented a reduction of the transitions during the simulations, while the SH3-SH2 deletion vanishes the Csk transitions, corroborating the experimental results mentioned and linking the conformational changes with the catalytic functionality of Csk. The study was complemented by the introduction of a known kinase inhibitor close to the Csk αC helix region where its consequences for the kinetic behavior and domain displacement of Csk were verified through detailed molecular dynamics. The findings describe the mechanisms involving the Csk αC helix for the transitions and also support the dynamic correlation between SH3 and SH2 domains against the Csk lobes and how local energetic restrictions or interactions in the Csk αC helix can play an important role for long-range motions. The results also allow speculation if the Csk activity is restricted to one specific conformation or a consequence of a state transition, this point being a target for future studies. However, the αC helix is revealed as a potential region for rational drug design.

Evolutionary Role of Water-Accessible Cavities in Src Homology 2 (SH2) Domains.

Protein excited states are fundamental in the understanding of biological function, despite the fact they are hardly observed using traditional biophysical methodologies. Pressure perturbation coupled with nuclear magnetic resonance (NMR) spectroscopy is a powerful physicochemical tool to glance at these low-populated high-energy states on a residue-by-residue basis and underpin mechanistic insights into protein functionalities. Here we performed pressure titrations using NMR spectroscopy and relaxation dispersion experiments to identify the low-lying energetic states of the c-Abl SH2 domain. By showing that the SH2 excited state contains a hydrated hydrophobic cavity, fast-exchange motions, and highly conserved residues facing the water-accessible hole, we discuss the implications of water-protein interactions in SH2 modules achieving high-affinity binding and promiscuous phospho-Tyr peptide recognition.

1H, 15N, and 13C resonance assignments of the SH3-like tandem domain of human KIN protein.

KIN is a DNA/RNA-binding protein conserved evolutionarily from yeast to humans and expressed ubiquitously in mammals. It is an essential nuclear protein involved in numerous cellular processes, such as DNA replication, class-switch recombination, cell cycle regulation, and response to UV or ionizing radiation-induced DNA damage. The C-terminal region of the human KIN (hKIN) protein is composed of an SH3-like tandem domain, which is crucial for the anti-proliferation effect of the full-length protein. Herein, we present the 1H, 15N, and 13C resonances assignment of the backbone and side chains for the SH3-like tandem domain of the hKIN protein, as well as the secondary structure prediction based on the assigned chemical shifts using TALOS-N software. This work prepares the ground for future studies of RNA-binding and backbone dynamics.

Novel STAT3 small-molecule inhibitors identified by structure-based virtual ligand screening incorporating SH2 domain flexibility.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain starting with short phosphotyrosylated peptides based on STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors, identified using structure-based virtual ligand screening (SB-VLS); while having favorable drug-like properties, suffer from weak binding affinities, possibly due to the high flexibility of the target domain. We conducted molecular dynamic (MD) simulations of the SH2 domain in complex with CJ-887, and used an averaged structure from this MD trajectory as an "induced-active site" receptor model for SB-VLS of 110,000 compounds within the SPEC database. Screening was followed by re-docking and re-scoring of the top 30% of hits, selection for hit compounds that directly interact with pY + 0 binding pocket residues R609 and S613, and testing for STAT3 targeting in vitro, which identified two lead hits with good activity and favorable drug-like properties. Unlike most small-molecule STAT3 inhibitors previously identified, which contain negatively-charged moieties that mediate binding to the pY + 0 binding pocket, these compounds are uncharged and likely will serve as better candidates for anti-STAT3 drug development. IMPLICATIONS: SB-VLS, using an averaged structure from molecular dynamics (MD) simulations of STAT3 SH2 domain in a complex with CJ-887, a known peptidomimetic binder, identify two highly potent, neutral, low-molecular weight STAT3-inhibitors with favorable drug-like properties.

Mutations in Hypervariable Domain of Venezuelan Equine Encephalitis Virus nsP3 Protein Differentially Affect Viral Replication.

Venezuelan equine encephalitis virus (VEEV) is one of the important human and animal pathogens. It forms replication enzyme complexes (RCs) containing viral nonstructural proteins (nsPs) that mediate the synthesis of virus-specific RNAs. The assembly and associated functions of RC also depend on the presence of a specific set of host proteins. Our study demonstrates that the hypervariable domain (HVD) of VEEV nsP3 interacts with the members of the FXR family of cellular proteins and also binds the Src homology 3 (SH3) domain-containing proteins CD2AP and SH3KBP1. Interactions with FXR family members are mediated by the C-terminal repeating peptide of HVD. A single short, minimal motif identified in this study is sufficient for driving efficient VEEV replication in the absence of HVD interactions with other host proteins. The SH3 domain-containing proteins bind to another fragment of VEEV HVD. They can promote viral replication in the absence of FXR-HVD interactions albeit less efficiently. VEEV replication can be also switched from an FXR-dependent to a chikungunya virus-specific, G3BP-dependent mode. The described modifications of VEEV HVD have a strong impact on viral replication in vitro and pathogenesis. Their effects on viral pathogenesis depend on mouse age and the genetic background of the virus.IMPORTANCE The replication of alphaviruses is determined by specific sets of cellular proteins, which mediate the assembly of viral replication complexes. Some of these critical host factors interact with the hypervariable domain (HVD) of alphavirus nsP3. In this study, we have explored binding sites of host proteins, which are specific partners of nsP3 HVD of Venezuelan equine encephalitis virus. We also define the roles of these interactions in viral replication both in vitro and in vivo A mechanistic understanding of the binding of CD2AP, SH3KBP1, and FXR protein family members to VEEV HVD uncovers important aspects of alphavirus evolution and determines new targets for the development of alphavirus-specific drugs and directions for viral attenuation and vaccine development.

Distinguishing Biomolecular Pathways and Metastable States.

Protein folding occurs in a high dimensional phase space, and the representation of the associated energy landscape is nontrivial. A widely applied approach to studying folding landscapes is to describe the dynamics along a small number of reaction coordinates. However, other strategies involve more elaborate analysis of the complex phase space. There have been many attempts to obtain a more detailed representation of all available conformations for a given system. In this work, we address this problem using a metric based on internal distances between amino acids to describe the differences between any two conformations. Using an effective projection method, we are able to go beyond the typical one-dimensional representation and provide intuitive two dimensional visualizations of the landscape. We refer to this method as the energy landscape visualization method (ELViM). We have applied this methodology using a Cα structure-based model to study the folding of two well-known proteins: SH3 domain and protein-A. Our visualization method yields a detailed description of the folding process, making possible the identification of transition state regions, and establishing the paths that lead to the native state. For SH3, we have analyzed structural differences in the distribution of folding routes. The competition between the native and mirror structures in protein A is also discussed. Finally, the method is applied to study conformational changes in the protein elongation factor thermally unstable. Distinct features of ELViM are that it does not require or assume a reaction coordinate, and it does not require analysis of kinetic aspects of the system.

The Ca2+ channel subunit CaV ß2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

ß-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

New Heteroleptic Ruthenium(II) Complexes with Sulfamethoxypyridazine and Diimines as Potential Antitumor Agents.

Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy)2smp](PF6) (1) and [Ru(phen)2smp](PF6) (2), in which smp = sulfamethoxypyridazine; bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1) or two phen (in 2) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine-one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes' preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.

NMR assignment of free 1H, 15N and 13C-Grb2-SH2 domain.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein composed of three domains, an N-terminal SH3 (nSH3), SH2 and a C-terminal SH3 (cSH3) domains. This multi-domain protein has been reported to be a key factor in many signaling pathways related to controlling cell survival, differentiation, and growth. The Grb2-SH2 domain has been a focus for the study of the interaction with peptides and small molecules to act as inhibitors in uncontrolled cell growth, and consequently inhibit tumor proliferation. Here we describe the almost complete assignment of the free SH2 domain at pH 7. This work prepares the ground for further structural studies, backbone dynamics, mapping of interactions and drug screening and development. TalosN secondary structure prediction showed great similarity with the available structures in the PDB.

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains.

BACKGROUND: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton's Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations. METHODS: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient. RESULTS: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes. CONCLUSIONS: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.

Small proline rich protein 2a in benign and malignant liver disease.

UNLABELLED: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis. CONCLUSION: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.

PTK2 and PTPN11 expression in myelodysplastic syndromes.

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors.

PTK2 and PTPN11 expression in myelodysplastic syndromes

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors. .

Breakdown of the Debye polarization ansatz at protein-water interfaces.

The topographical and physico-chemical complexity of protein-water interfaces scales down to the sub-nanoscale range. At this level of confinement, we demonstrate that the dielectric structure of interfacial water entails a breakdown of the Debye ansatz that postulates the alignment of polarization with the protein electrostatic field. The tendencies to promote anomalous polarization are determined for each residue type and a particular kind of structural defect is shown to provide the predominant causal context.

A novel missense mutation in the SH2 domain of the STAT5B gene results in a transcriptionally inactive STAT5b associated with severe IGF-I deficiency, immune dysfunction, and lack of pulmonary disease.

CONTEXT: Signal transducer and activator of transcription 5b (STAT5b) deficiency, first reported in a patient who carried a p.Ala630Pro missense mutation in the Src homology 2 (SH2) domain, results in a rare clinical condition of GH insensitivity (GHI), IGF-I deficiency (IGFD), and severe immune dysregulation manifesting as progressive worsening of pulmonary function. PATIENT: The new patient presented with severe cutaneous eczema, episodic infections in the first years of life, and autoimmune thyroiditis. Immunological evaluation revealed T lymphopenia, but severe pulmonary symptoms were notably absent. She concomitantly exhibited pronounced growth failure, reaching an adult height of 124.7 cm [-5.90 SD score (SDS)]. Endocrine evaluations (normal provocative GH tests; low serum IGF-I, -3.7 SDS, and IGF-binding protein-3, -4.5 SDS) were consistent with GHI and IGFD. RESULTS: Analysis of the STAT5B gene revealed a novel homozygous missense mutation, p.Phe646Ser, located within the ßD' strand of the SH2 domain. Reconstitution studies demonstrated expression of the p.Phe646Ser variant was less robust than wild type but, in contrast to the previously described STAT5B p.Ala630Pro SH2 mutation, could be phosphorylated in response to GH and interferon-γ. The phosphorylated p.Phe646Ser, however, could not drive transcription. CONCLUSION: A novel STAT5B p.Phe646Ser mutation has been identified in a patient with clinical characteristics of STAT5b deficiency. Only the second STAT5B missense mutation identified, its lack of transcriptional activities despite GH-induced phosphorylation, confirms the crucial role of STAT5b for regulating the expression of IGF1 and provides insights into the importance of the SH2 ßD' strand for full STAT5b transcriptional activities. Whether the phosphorylated p.Phe646Ser variant retained functions that prevented pulmonary distress remains unresolved.

Activation of c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration by hexachlorobenzene in MDA-MB-231 human breast cancer cell line.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR) protein. HCB is a tumor cocarcinogen in rat mammary gland and an inducer of cell proliferation and c-Src kinase activity in MCF-7 breast cancer cells. This study was carried out to investigate HCB action on c-Src and the human epidermal growth factor receptor (HER1) activities and their downstream signaling pathways, Akt, extracellular-signal-regulated kinase (ERK1/2), and signal transducers and activators of transcription (STAT) 5b, as well as on cell migration in a human breast cancer cell line, MDA-MB-231. We also investigated whether the AhR is involved in HCB-induced effects. We have demonstrated that HCB (0.05µM) produces an early increase of Y416-c-Src, Y845-HER1, Y699-STAT5b, and ERK1/2 phosphorylation. Moreover, our results have shown that the pesticide (15 min) activates these pathways in a dose-dependent manner (0.005, 0.05, 0.5, and 5µM). In contrast, HCB does not alter T308-Akt activation. Pretreatment with a specific inhibitor for c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine [PP2]) prevents Y845-HER1 and Y699-STAT5b phosphorylation. AG1478, a specific HER1 inhibitor, abrogates HCB-induced STAT5b and ERK1/2 activation, whereas 4,7-orthophenanthroline and α-naphthoflavone, two AhR antagonists, prevent HCB-induced STAT5b and ERK1/2 phosphorylation. HCB enhances cell migration evaluated by scratch motility and transwell assays. Pretreatment with PP2, AG1478, and 4,7-orthophenanthroline suppresses HCB-induced cell migration. These results demonstrate that HCB stimulates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways in MDA-MB-231. c-Src, HER1, and AhR are involved in HCB-induced increase in cell migration. The present study makes a significant contribution to the molecular mechanism of action of HCB in mammary carcinogenesis.

NFATc1 and TNFalpha expression in giant cell lesions of the jaws.

BACKGROUND: Activation mutations of SH3BP2 gene have been demonstrated in cherubism and central giant cell lesion (CGCL). In the present study we first attempted to investigate the SH3BP2 gene in peripheral giant cell lesion (PGCL). The effect of SH3BP2 gene mutations on the transcription of the downstream genes nuclear factor of activated T cells (NFATc1) and the cytokine tumor necrosis factor-alpha (TNF-alpha) was also investigated together with the immunolocalization of NFATc1 protein in a set of cases of PGCL, CGCL and cherubism with and without SH3BP2 mutation. METHOD: Fresh samples of five PGCL, five CGCL and one cherubism cases were included in this study. One of the samples of CGCL presented a somatic heterozygous mutation c.1442A>T in exon 11. The cherubism case showed a heterozygotic substitution c.320C>T in both blood and lesion. These mutations were previously published. All coding and flanking regions of the SH3BP2 gene were sequenced in the cases of PGCL. The real-time polymerase chain reaction (RT-PCR) was performed to analyze the transcription of NFATc1 and TNF-alpha genes. The immunohistochemical analysis of the NFATc1 protein was also performed. RESULTS: No SH3BP2 gene mutation was found in PGCL. The RT-PCR showed increased expression of NFATc1 and decreased transcription of TNF-alpha in all the samples. The immunohistochemical analysis of the NFATc1 protein showed a predominant nuclear staining in the multinucleated giant cells. CONCLUSION: The development of giant cells lesions of the jaws and cherubism are possibly mediated by overexpression of NFAT in the nucleus of the multinucleated cells.

Novel mutations in the SH3BP2 gene associated with sporadic central giant cell lesions and cherubism.

Central giant cell lesion (CGCL) is a reactive bone lesion that occurs mainly in the mandible, characterized by the multinucleated osteoclast-like giant cells in a background of oval to spindle-shaped mononuclear cells. The etiology is unknown and occurs more commonly in young adults. Cherubism, a rare disease found predominantly in females has histologic characteristics indistinguishable from those of CGCL and is caused by mutations mostly present in exon 9 of the SH3BP2 gene. In this study, we investigated four cases of CGCL and one case of cherubism. DNA was extracted from peripheral blood and tumor tissue and all coding and flanking regions of the SH3BP2 amplified by PCR and directly sequenced to identify underlying mutations. Two novel mutations were found; a heterozygous missense mutation c.1442A>T (Q481L) in exon 11 in one sporadic case of CGCL and a heterozygous germline and tumor tissue missense mutation c.320C>T (T107M) in exon 4 in one patient with cherubism. These findings open a new window to investigate the possible relationship between the pathogenesis of the cherubism and CGCL.