Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

New insights into the link between cardiovascular disease and depression.

INTRODUCTION: Although the association between depression and cardiovascular disease (CVD) is well documented, the underlying mechanisms for this relationship remain unclear. In this paper, we present three possible models which account for the comorbidity between depression and cardiovascular disease. MODELS: The first model outlines depression as a risk factor for CVD and the second model presents CVD as a risk factor for depression. The third model proposes a common underlying pathway related to the effects of chronic stress on the body in manifesting as depression or cardiovascular disease. CONCLUSIONS: If the proposed model holds true, it may be possible that an intervention initiated before overt manifestations of CVD or depression become apparent, may delay or prevent the onset of these serious clinical entities.

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements.

Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr452 and Tyr474 are polarised in both fragments while Tyr497 is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr474 polarization while the polarization of Tyr452 is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr474 polarization is not due to protein-protein contacts but to interaction with DNA.

1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.

Solution structure of the glucocorticoid receptor DNA-binding domain.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.

Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA-binding domain.

We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism.

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.

High level expression in Escherichia coli of the DNA-binding domain of the glucocorticoid receptor in a functional form utilizing domain-specific cleavage of a fusion protein.

A fragment comprising the DNA-binding domain of the human glucocorticoid receptor has been expressed in a functional form in Escherichia coli as a fusion protein with protein A from Staphylococcus aureus. The DNA-binding domain was purified to apparent homogeneity by affinity chromatography on IgG-Sepharose and DNA-cellulose, a purification scheme which does not involve denaturation of the protein at any step. The DNA-binding domain was separated from the protein A part of the fusion protein by domain-specific enzymatic cleavage with chymotrypsin while immobilized on IgG-Sepharose. The recombinant protein has been characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reactivity to iodoacetate and was found to correspond to the primary structure derived from the cDNA sequence. DNase I footprinting showed that the purified recombinant protein bound to the same DNA sequences on the mouse mammary tumor virus long terminal repeat as glucocorticoid receptor purified from rat liver does. About 10 times more recombinant protein, on a molar basis, was needed to obtain the same level of protection. However, the protection of the three different footprints (1.3, 1.4, and 1.5') by the recombinant protein differed greatly from that of the natural receptor, with virtually no protection of footprint 1.4. This indicates cooperative binding of the natural receptor to adjacent footprints, dependent on other regions of the receptor than the DNA-binding domain.

DNA binding of glucocorticoid receptor protein A fusion proteins expressed in E. coli.

In an effort to obtain large quantities of glucocorticoid receptor (GR) protein for functional and structural studies, several truncated versions of the human glucocorticoid receptor (hGR) have been expressed in E. coli as C-terminal fusion proteins with protein A. The amount of expressed protein was between 5 and 25 mg/l in the culture. South-Western blotting was initially used to demonstrate the DNA binding capacity of fusion proteins containing the DNA binding domain of GR. The hybrid proteins were highly enriched in the insoluble fraction after cell lysis. For further purification and characterization the fusion proteins were solubilized in 8 M urea. The concentration of denaturing agent was reduced by dilution and the fusion proteins were allowed to refold. The renatured GR protein A fusion proteins bound to DNA in a nitrocellulose filter binding assay. We also show that it is possible to purify the renatured fusion protein to apparent homogeneity using a single chromatographic step on DNA-cellulose.

Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation.

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.