Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding

The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Free fatty acid receptors:structural models and elucidation of ligand binding interactions

BACKGROUND: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators.

RESULTS: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators.

CONCLUSIONS: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

Chromatin binding by the androgen receptor in prostate cancer

Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.

METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.

RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.

CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization

AMPA receptors are tetrameric glutamate-gated ion channels that mediate fast synaptic neurotransmission in mammalian brain. Their subunits contain a two-lobed N-terminal domain (NTD) that comprises over 40% of the mature polypeptide. The NTD is not obligatory for the assembly of tetrameric receptors, and its functional role is still unclear. By analyzing full-length and NTD-deleted GluA1-4 AMPA receptors expressed in HEK 293 cells, we found that the removal of the NTD leads to a significant reduction in receptor transport to the plasma membrane, a higher steady state-to-peak current ratio of glutamate responses, and strongly increased sensitivity to glutamate toxicity in cell culture. Further analyses showed that NTD-deleted receptors display both a slower onset of desensitization and a faster recovery from desensitization of agonist responses. Our results indicate that the NTD promotes the biosynthetic maturation of AMPA receptors and, for membrane-expressed channels, enhances the stability of the desensitized state. Moreover, these findings suggest that interactions of the NTD with extracellular/synaptic ligands may be able to fine-tune AMPA receptor-mediated responses, in analogy with the allosteric regulatory role demonstrated for the NTD of NMDA receptors.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

The structure of arabidopsis NPR1 : its function as a salicylic acid receptor and a metal-binding protein

The Arabidopsis NPRI protein regulates systemic acquired resistance dependent on salicylic acid. Analyses by plant two-hybrid analysis in vivo and pull-down assays in vitro showed that the BTB/POZ domain of NPRI at the N-terminus serves as an autoinhibitory domain to negate the function of the transactivation domain at the C-terminus through direct binding of these two domains. I t was also shown that the binding of the BTB/POZ domain to the C-terminus of NPRI was abolished by SA treatment, suggesting that SA could interfere directly with this binding. By gel filtration, it was demonstrated that SA affects the conformation of full-length NPRl , confirming the role of NPRI as an SA receptor. Gel filtration analysis also indicated that NPRI could be converted from an oligomer to a dimer with SA treatment. Furthermore, one N-terminal deletion ~513 has been shown to act as a metal-binding protein and its two Cys-521 and Cys-529 are important for binding to Ni 2 + by pull-down assays.

Predicting the Pose of β-Casomorphin-5 and 7 in the Opioid Receptors

The opioid receptors consist of three main subtypes; μ, δ, and κ. Previous binding studies have shown that fragments of the milk protein, β-casein, known as β-casomorphins are agonists of these receptors which are selective for the μ receptor subtype. Using the crystal structures of these three receptors, computational molecular docking studies were done using the software GOLD to determine the conformation of β-casomorphin-5 and 7 when they bind to these three opioid receptors. GOLD was able to discriminate among the three receptors when docking the rigid ligands co-crystalized with the receptors. However, GOLD could not discriminate among the three receptors for either of the highly flexible β-casomorphins. A per amino acid scoring method was developed to overcome this problem. This method was used to predict the conformation of both β-casomorphin-5 and 7 in the μ receptor and determine that the two amino acid residues, Lys303 and Trp318 of the μ receptor are responsible for discriminating among the three receptor subtypes for binding of the β-casomorphin-5 and 7.