Hexapeptide fragment of carcinoembryonic antigen which acts as an agonist of heterogeneous ribonucleoprotein M.

Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH2 (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations.

Expression of leukemia/lymphoma-related factor (LRF/POKEMON) in human breast carcinoma and other cancers.

The POK family of proteins plays an important role in not only embryonic development and cell differentiation, but also in oncogenesis. Leukemia/lymphoma-related factor (LRF) belongs to the POK family of transcriptional repressors and is also known as POK erythroid myeloid ontogenic factor (POKEMON), which binds to short transcripts of HIV-1 (FBI-1) and TTF-1 interacting peptide (TIP21). Its oncogenic role is known only in lymphoma, non-small cell lung carcinoma, and malignant gliomas. The functional expression of LRF in human breast carcinoma has not yet been confirmed. The aim of this study was to investigate and compare the expression of LRF in human breast cancer tissues and other human tumors. The expression of LRF mRNA transcripts and protein was observed in twenty human benign and malignant breast biopsy tissues. Expression of LRF was observed in several formalin-fixed tissues by immunohistochemistry and immunofluorescence. All malignant breast tissues expressed mRNA transcripts and protein for LRF. However, 40% and 15% benign breast biopsy tissues expressed LRF mRNA transcripts and protein, respectively. The overall expression of LRF mRNA transcripts and total protein was significantly more in malignant breast tissues than the benign breast tissues. LRF expression was also observed in the nuclei of human colon, renal, lung, hepatocellular carcinomas and thymoma tumor cells. In general, a significantly higher expression of LRF was seen in malignant tissues than in the corresponding benign or normal tissue. Further studies are warranted to determine the malignant role of LRF in human breast carcinoma.

Interaction of tgf-beta with immune cells in airway disease.

Asthma, chronic obstructive pulmonary disorder (COPD), and cystic fibrosis (CF), chronic diseases of the airways, are characterized by symptoms such as inflammation of the lung tissue, mucus hypersecretion, constriction of the airways, and excessive fibrosis of airway tissue. Transforming growth factor (TGF)-beta, a cytokine that affects many different cell processes, has an important role in the lungs of patients with some of these chronic airway diseases, especially with respect to airway remodeling. Eosinophils can be activated by and are a major source of TGF-beta in asthma. The action of TGF-beta also shows associations with other cell types, such as T cells and neutrophils, which are involved in the pathogenesis of asthma. TGF-beta can perpetuate the pathogenesis of COPD and CF, as well, through its induction of inflammation via release from and action on different cells. The intracellular signaling induced by TGF-beta in various cell types has been elucidated and may point to mechanisms of action by TGF-beta on different structural or immune cells in these airway diseases. Some possible treatments, especially that prevent the deleterious airway changes induced by the action of either eosinophils or TGF-beta in asthma, have been investigated. TGF-beta-induced signaling pathways, especially those in different cell types in asthma, COPD, or CF, may provide potential therapeutic targets for the treatment of some of the most devastating airway diseases.

Calculation of weakly polar interaction energies in polypeptides using density functional and local Møller-Plesset perturbation theory.

The interaction energies of ubiquitous weakly polar interactions in proteins are comparable with those of hydrogen bonds, consequently, they stabilize local, secondary, and tertiary structures. However, the most widely-used density functionals fail to describe the weakly polar interactions. Thus, it is important to find and test functionals which adequately describe and quantify the energetics of such interactions. For this purpose, interaction energies in the hydrophobic core of rubredoxin (PDB id: 1rb9) and in the S22 subset of the JSCH-2005 benchmark database were computed with the BHandHLYP and PWPW91 functionals and with the pseudospectral implementation of the local MP2 (PS-LMP2) method. The cc-pVDZ, cc-pVTZ(-f), cc-pVTZ, cc-pVQZ(-g), aug-cc-pVDZ, aug-cc-VTZ(-f), and aug-cc-pVTZ basis sets were used for the calculations. In the S22 subset the PS-LMP2 results were extrapolated to the complete basis set limit. Furthermore, the a posteriori counterpoise method of Boys and Bernardi was used to correct the basis set superposition errors in the calculation of interaction energies. Calculations using the BHandHLYP functional, both for the various weakly polar interactions in rubredoxin and for the dispersion interactions in the S22 subset, were in good agreement with those using the coupled cluster (CCSD(T)) and the resolution of identity MP2 (RIMP2) methods and clearly outperformed both the PWPW91 functional and the PS-LMP2 method. The results for the S22 hydrogen bonded subset, obtained with PWPW91 calculations, were closest to those of the reference high level calculations. For the "mixed" (hydrogen bonded and dispersive) interactions in the S22 subset, results obtained with the BHandHLYP and PS-LMP2 calculations agreed well with the reference calculations.

Development of glycyl radical parameters for the OPLS-AA/L force field.

On the basis of quantum chemical calculations C(alpha)-glycyl radical parameters have been developed for the OPLS-AA/L force field. The molecular mechanics hypersurface was fitted to the calculated quantum chemical surface by minimizing their molecular mechanics parameter dependent sum-of-squares deviations. To do this, a computer program in which the molecular mechanics energy derivatives with respect to the parameters were calculated analytically was developed, implementing the general method of Lifson and Warshel (J Chem Phys 1968, 49, 5116) for force field parameter optimization. This program, in principle, can determine the optimal parameter set in one calculation if enough representative value points on the quantum chemical potential energy surface are available and there is no linear dependency between the parameters. Some of the parameters in quantum calculations, including several new torsion types around a bond as well as angle parameters at a new central atom type, are not completely separable. Consequently, some restrictions and/or presumptions were necessary during parameter optimization. The relative OPLS-AA energies reproduced those calculated quantum chemically almost perfectly.

The effect of electron correlation on the conformational space of melatonin.

The pineal gland hormone melatonin regulates several physiological processes including circadian rhythm and also alleviates oxidative stress-induced degenerative diseases. In spite of its important biological roles, no high level ab initio conformational study has been conducted to reveal its structural features. In this work, the conformational flexibility of melatonin was investigated using correlated ab initio calculations. Conformers, obtained previously at the Hartree-Fock level (HF/6-31G*), were fully optimized using second order Møller-Plesset perturbation theory applying the frozen core approximation (MP2(FC)/6-31G*). Furthermore, single-point MP4(SDQ,FC)/6-31G*//MP2(FC)/6-31G* computations were performed to investigate the effect of higher order perturbation terms. The HF and MP2 conformational spaces are considerably different: the initial 128 structures converged into 102 different local minima as confirmed by frequency calculations; 28 new minima appeared and 26 previous HF local minima disappeared; no "all-trans" C3 side chain conformations are seen at the MP2(FC) level. The MP2 global minimum conformation is stabilized by an aromatic-side chain interaction.

Quantum chemical quantification of weakly polar interaction energies in the TC5b miniprotein.

The tertiary structure of the TC5b miniprotein is stabilized by inter-residue interactions of the Trp-cage, which is composed of a Tyr and several Pro residues surrounding a central Trp residue. The interactions include Ar-Ar (aromatic side-chain-aromatic side-chain), Ar-NH (aromatic side-chain-backbone amide), and CH-pi (aromatic side-chain-aliphatic hydrogen) interactions. In the present work, the strength of the weakly polar interactions found in the TC5b miniprotein was quantified using all of the available 38 NMR structures (1L2Y) from the Protein Data Bank with DFT quantum chemical calculations at the BHandHLYP/cc-pVTZ level of theory and molecular fragmentation with capping of the partial structures. The energies of interaction between the individual residues of the Trp-cage range between -5.85+/-1.41 and -21.30+/-0.88 kcal mol(-1), leading to a significant total structural stabilization energy of -52.13+/-2.56 kcal mol(-1) of which about 50% is from the weakly polar interactions. Furthermore, the strengths of the individual weakly polar interactions are between -2.32+/-0.17 and -2.93+/-0.12 kcal mol(-1) for the CH-pi interactions, between -2.48+/-0.97 and -3.09+/-1.02 kcal mol(-1) for the Ar-NH interaction and -2.74+/-1.06 kcal mol(-1) for the Ar-Ar interaction.

On the role of transforming growth factor-beta in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells.

BACKGROUND: Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-beta2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-beta in retinoic acid-mediated growth inhibition in pancreatic cancer cells. RESULTS: Retinoic acid markedly inhibited proliferation of two cell lines (Capan-2 and Hs766T) in a concentration and time-dependent manner. Retinoic acid increased TGF-beta2 mRNA content and secretion of the active and latent forms of TGF-beta2 (measured by ELISA and bioassay). The concentrations of active and TGF-beta2 secreted in response to 0.1 - 10 muM retinoic acid were between 1-5 pM. TGF-beta2 concentrations within this range also inhibited proliferation. A TGF-beta neutralizing antibody blocked the growth inhibitory effects of retinoic acid in Capan-2 cells and partially inhibitory the effects in Hs766T cells. CONCLUSION: These findings indicate that TGF-beta can cause growth inhibition of pancreatic cancer cells, in a p53-independent manner. Furthermore, it demonstrates the fundamental role of TGF-beta in growth inhibition in response to retinoic acid treatment is preserved in vitro.

Decoding IgE Fc receptors.

Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases by interacting with two membrane receptors: high-affinity FcepsilonRI and low-affinity FcepsilonRII (CD23). Allergeninduced IgE-occupied FcepsilonRI aggregation on the mast cell or basophil cell surface leads to the activation of intracellular signaling events and eventually the release of pre-formed and de novo synthesized inflammatory mediators. The role of FcepsilonRII in allergic diseases has been proposed to include regulation of IgE synthesis, enhanced histamine release from basophils, and a contribution to Ag-IgE complex presentation but the exact function of CD23 remains poorly understood. This review summarizes some new developments in IgE Fc-receptor studies with an emphasis on regulation of FcepsilonRI expression and signal transduction, including monomeric IgE, lipid raft segregation, and some recently identified negative regulators. A better understanding of signaling events following IgE FcR aggregation will shed new light on how allergy patients might be treated more safely and effectively.

Gastrin 1-6 promotes growth of colon cancer cells through non-CCK receptors.

Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.

Conformational analysis of Ac-NPGQ-NH(2) and Ac-VPaH-NH(2) by vibrational circular dichroism spectroscopy combined with molecular dynamics and quantum chemical calculations.

Conformational properties of two potentially beta-turn forming peptides were determined using a strategy which combines MD simulations, IR and VCD spectroscopy and quantum chemical calculations. This strategy could be a useful alternative for solution conformational analysis of short flexible peptides and could help to identify VCD features which are as yet unknown.

Immunomodulatory role of vascular endothelial growth factor and angiopoietin-1 in airway remodeling.

The blood vessels formed in asthmatic airways are involved in inflammatory and airway remodeling processes in chronic asthma. Vascular endothelial cell growth factor (VEGF) and angiopoietin-1 (Ang-1) are primary angiogenic growth factors, involved in the formation of such blood vessels. VEGF has been reported to contribute to non-specific airway hyper-responsiveness, have chemotactic effects on eosinophils, and enhance airway smooth muscle cell proliferation. Furthermore, Th2 cells have receptors for VEGF, and Th2-associated cytokines increase VEGF production. There are reports that elevated levels of VEGF correlates with the severity of asthma. Ang-1 has been shown to induce pro-inflammatory effects such as eosinophil chemotaxis via tie-2 receptors. Reports indicate ang-1 contribution to increased secretion of matrix metalloproteinase-2 (MMP-2) and decreased secretion of tissue inhibitors of metalloproteinase-2 (TIMP-2). However, Ang-1 has also been shown to exhibit several anti-inflammatory properties such as suppressing expression of adhesion molecules, blocking vascular permeability and eosinophil chemotaxis induced by VEGF. These findings support the notion that apart from their roles in blood vessels formation, these angiogenic growth factors are directly involved in the pathogenesis of chronic asthma. This paper reviews individual and combined roles of VEGF and Ang-1. The potential therapeutic applications involving these factors are also discussed.

Following the TRAIL to apoptosis.

Apoptosis, programmed cell death, eliminates injured or harmful cells. It can mediate its response through the actions of death ligands including TRAIL. TRAIL, a member of TNF superfamily, induces apoptosis of transformed cells through the action of death domain receptors DR-4 and DR5. It directly induces apoptosis through an extrinsic pathway, which involves the activation of caspases. TRAIL also is able to prevent apoptosis through the actions of its decoy receptors DcR-1 and DcR-2. Various regulators of TRAIL include FADD, IAPs, Bcl-2s, p53, and FLIPs. TRAIL is present in cells involved in asthma including eosinophils, mast cells, fibroblasts, and airway epithelial cells. It is expressed in airway remodeling and may be linked with the pathways of transforming growth factor-beta1, which is thought to cause damage to the epithelium. The repair process of the epithelium is hindered as a result of increased apoptosis induced by TGF-beta1, which overlaps with the pathways of TRAIL. Analogs of TRAIL could have therapeutical applications for asthma. TRAIL is also seen as the basis for a "miracle" drug for cancer because of its ability to selectively kill cancer cells.

Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors.

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.

Importance of the central region of lamprey gonadotropin-releasing hormone III in the inhibition of breast cancer cell growth.

Naturally occurring isoforms of the decapeptide gonadotropin-releasing hormone (GnRH) share residues 1-4 and 9-10. lGnRH-III, the third isoform isolated in the sea lamprey has no endocrine effect in mammals but shows a direct antiproliferative effect on human breast, prostate and endometrial cancer cell lines. To investigate these features, residues 5-8 of lGnRH-III were systematically replaced with Ala. The ability of the synthetic analogs to interact with receptors on MDA-MB 231 human breast cancer cells and their effect on the growth of the same cell line were investigated. [Ala6]lGnRH-III and [Ala7]lGnRH-III have neither receptor binding nor antiproliferative activity. Replacement of His5 with Ala resulted in an analog that binds to the receptor but does not have antiproliferative activity. The results are in agreement with previous reports that modifications of Lys at position 8 are well tolerated.

High and low affinity receptors mediate growth effects of gastrin and gastrin-Gly on DLD-1 human colonic carcinoma cells.

Gastrin (G17) and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these effects is controversial. A recent study demonstrated the presence of a low affinity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth-promoting effects of gastrin peptides. Binding of [Leu(15)]G17 and [Leu(15)]G17-Gly to DLD-1 cell membranes in competition with [(3)H]G17-Gly was examined. Binding of [(3)H]cholecystokinin-8 (CCK8) to DLD-1 cell membranes was also assessed. Whole cell binding experiments were carried out using [(125)I-Tyr(12),Leu(15)]G17-Gly. In addition, the ability of [Leu(15)]G17 and [Leu(15)]G17-Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu(15)]G17 and [Leu(15)]G17-Gly competed with [(3)H]G17-Gly at both a high and a low affinity site on DLD-1 membranes. The IC(50) values for [Leu(15)]G17 were 6.0 x 10(-8) M and 6.9 x 10(-6) M while those for [Leu(15)]G17-Gly were 3.2 x 10(-9) M and 4.9 x 10(-6) M. [(3)H]CCK8 did not bind to either site. [Leu(15)]G17-Gly also competed with [(125)I-Tyr(12),Leu(15)]G17-Gly at both a high and a low affinity site on DLD-1 cells with similar affinities as observed with membranes. [Leu(15)]G17 and [Leu(15)]G17-Gly significantly stimulated the growth of DLD-1 cells in a dose-dependent and biphasic manner. The binding profiles of the peptides tested suggest that these sites are different from previously identified wild-type and mutant CCK(1) or CCK(2) receptors.

A specific binding site for a fragment of the B-loop of epidermal growth factor and related peptides.

Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125I-EGF and [3'4'-3H-Tyr(22,29), Abu(20,31)]EGF(20-31)-NH(2). Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity.