We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.
Aberrant glycosylation is a universal feature of cancer cells, and certain glycan structures are well-known markers for tumor progression. Availability and composition of sugars in the microenvironment may affect cell glycosylation. Recent studies of human breast tumor cell lines indicate their ability to take up and utilize fructose. Here we tested the hypothesis that adding fructose to culture as a carbon source induces phenotypic changes in cultured human breast tumor cells that are associated with metastatic disease. MDA-MB-468 cells were adapted to culture media in which fructose was substituted for glucose. Changes in cell surface glycan structures, expression of genes related to glycan assembly, cytoskeleton F-actin, migration, adhesion and invasion were determined. Cells cultured in fructose expressed distinct cell-surface glycans. The addition of fructose affected sialylation and fucosylation patterns. Fructose feeding also increased binding of leukoagglutinating Phaseolus vulgaris isolectin, suggesting a possible rise in expression of branching β-1, 6 GlcNAc structures. Rhodamine-phalloidin staining revealed an altered F-actin cytoskeletal system. Fructose accelerated cellular migration and increased invasion. These data suggest that changing the carbon source of the less aggressive MDA-MB-468 cell line induced characteristics associated with more aggressive phenotypes. These data could be of fundamental importance due to the markedly increased consumption of sweeteners containing free fructose in recent years, as they suggest that the presence of fructose in nutritional micro-environment of tumor cells may negatively affect the outcome for some breast cancer patients.
A set of novel tachykinin-like peptides has been isolated from bullfrog brain and gut. These compounds, ranatachykinin A (RTKA), ranatachykinin B (RTKB), and ranatachykinin C (RTKC), were named for their source, Rana catesbeiana, and their homology to the tachykinin peptide family. We present the first report of the micelle-bound structures and pharmacological actions of the RTKs. Generation of three-dimensional structures of the RTKs in a membrane-model environment using (1)H NMR chemical shift assignments, two-dimensional NMR techniques, and molecular dynamics and simulated annealing procedures allowed for the determination of possible prebinding ligand conformations. RTKA, RTKB, and RTKC were determined to be helical from the midregion to the C-terminus (residues 4-10), with a large degree of flexibility in the N-terminus and minor dynamic fraying at the end of the C-terminus. The pharmacological effects of the RTKs were studied by measuring the elevation of intracellular Ca(2+) in Chinese hamster ovarian cells stably transfected with the bullfrog substance P receptor (bfSPR). All of the RTKs tested elicited Ca(2+) elevations with a rank order of maximal effect of RTKA >/= SP > RTKC >/= RTKB. A high concentration (1 microM) of the neuropeptides produced varying degrees of desensitization to a subsequent challenge with the same or different peptide, while a low concentration (1 pM) produced sensitization at the bfSPR. Our data suggest differences in amino acid side chains and their charged states at the C-terminal sequence or differences in secondary structure at the N-terminus, which do not overlap according to the findings in this paper, may explain the differing degree and type of receptor activation seen at the bfSPR.
Expression of sialyl Lewis x (sLe x ) and sLe a on tumor cells is thought to facilitate metastasis by promoting cell adhesion to selectins on vascular endothelial cells. Experiments supporting this concept usually bypass the early steps of the metastatic process by employing tumor cells that are injected directly into the blood. We investigated the relative role of sLe x oligosaccharide in the dissemination of breast carcinoma, employing a spontaneous murine metastasis model. An sLe x deficient subpopulation of the 4T1 mammary carcinoma cell line was produced by negative selection using the sLe x -reactive KM93 MAb. This subpopulation was negative for E-selectin binding but retained P-selectin binding. Both sLe x -negative and -positive cells grew at the same rate; however, sLe x -negative cells spread more efficiently on plates and had greater motility in wound-scratch assays. Mice inoculated in the mammary fat pad with sLe x -negative and -positive variants produced lung metastases. However, the number of lung metastases was significantly increased in the group inoculated with the sLe x -negative variant (p 5 0.0031), indicating that negative selection for the sLe x epitope resulted in enrichment for a subpopulation of cells with a high metastatic phenotype. Cell variants demonstrated significant differences in cellular morphology and pattern of tumor growth in primary and secondary tumor sites. These results strongly suggest that loss of sLe x may facilitate the metastatic process by contributing to escape from the primary tumor mass. ' 2005 Wiley-Liss, Inc.Key words: sialyl Lewis x antigen; metastasis; 4T1 cells; breast cancer Tumor metastasis is a multistep process requiring detachment of malignant cells from the primary tumor, penetration of blood or lymph vessels, attachment to endothelium of distant organs and formation of new tumor foci. 1,2 Tissue invasion and metastasis of tumor cells are highly dependent on cell-cell interactions, many of which involve alterations in cell surface glycosylation patterns. 3,4 Although adhesion pathways utilized by tumor cells show considerable diversity, members of the selectin family of molecules and numerous neolactoseries antigens highly expressed on the tumor cell surface are involved in tumor metastasis by mediating binding of blood-borne tumor cells via E-and/or P-selectin to vascular endothelium. [5][6][7][8][9][10] Much of what we know about selectin interactions comes from studies of leukocytes. 11,12 Functionally, the binding of selectins to leukocytes requires sialylated and fucosylated carbohydrate structures; their prototypes are SAa2-3Galb1-4(Fuca1-3)GlcNAc and SAa2-3Galb1-3(Fuca1-4)GlcNAc, referred to as sLe x and sLe a , respectively. [13][14][15] Both sLe x and sLe a are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. [16][17][18][19] These determinants not only are markers for cancer but also are functionally implicated ...
The mechanism by which peptides bind to micelles is believed to be a two-phase process, involving (i). initial electrostatic interactions between the peptide and micelle surface, followed by (ii). hydrophobic interactions between peptide side chains and the micelle core. To better characterize the electrostatic portion of this process, a series of pulse field gradient nuclear magnetic resonance (PFG-NMR) spectroscopic experiments were conducted on a group of neuropeptides with varying net cationic charges (+1 to +3) and charge location to determine both their diffusion coefficients and partition coefficients when in the presence of detergent micelles. Two types of micelles were chosen for the study, namely anionic sodium dodecylsulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles. Results obtained from this investigation indicate that in the case of the anionic SDS micelles, peptides with a larger net positive charge bind to a greater extent than those with a lesser net positive charge (bradykinin > substance P > neurokinin A > Met-enkephalin). In contrast, when in the presence of zwitterionic DPC micelles, the degree of mixed-charge nature of the peptide affects binding (neurokinin A > substance P > Met-enkephalin > bradykinin). Partition coefficients between the peptides and the micelles follow similar trends for both micelle types. Diffusion coefficients for the peptides in SDS micelles, when ranked from largest to smallest, follow a trend where increasing net positive charge results in the smallest diffusion coefficient: Met-enkephalin > neurokinin A > bradykinin > substance P. Diffusion coefficients when in the presence of DPC micelles, when ranked from largest to smallest, follow a trend where the presence of negatively-charged side chains results in the smallest diffusion coefficient: bradykinin > Met-enkephalin > substance P > neurokinin A.
It is well known that neuropeptides interact with lipid vesicles in a manner similar to biological membranes, with electrostatic interactions between the two providing a mechanism for concentrating the peptide at the vesicle's surface, followed by hydrophobic interactions between the peptide and the core of the vesicle that induce and stabilize secondary structure motifs. In an effort to understand these interactions to a greater extent, our group has developed a series of anionic micelles (SDS) containing various concentrations of the bile salt CHAPS, which is used as a model for cholesterol. The incorporation of CHAPS into the hydrophobic core of these micelles should alter the degree to which the neuropeptide can insert itself, affecting structure. These interactions were investigated using two‐dimensional NMR, pulse‐field gradient (PFG) NMR, and molecular modeling experiments. The results of this study clearly indicate that electrostatic and hydrophobic interactions between the micelle and neuropeptide are completely independent of one another. Increasing the concentration of CHAPS to 15 mM in the micelles blocks the insertion of the hydrophobic side chains of the neuropeptide into the hydrophobic core of the micelles. The electrostatic interactions as determined by diffusion measurements are not affected by the presence of increasing CHAPS concentration. Our observations are consistent with the predictions of Seelig (A. Seelig and J. Seelig, “Interaction of Drugs and Peptides with the Lipid Membrane,” in Structure and Function of 7TM Receptors, T. W. Schwartz, S. A. Hjorth, and T. S. Kastrup, Eds., Munksgaard: Location, 1996). © 2001 John Wiley & Sons, Inc. Biopolymers 58: 593–605, 2001
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