A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental and biomedical effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ERs) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions, and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate and deleterious to health signaling) may be better controlled. The fractal approach is applied to analyte–ER interaction data available in the literature. The units for the different parameters (rate coefficients and affinities) in fractal-type kinetics are different from those obtained in classical kinetics. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D_f) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. This has biomedical and environmental implications in that the dissociation (and the binding) rate coefficient may be used to alleviate (deleterious effects) or enhance (beneficial effects) by selective modulation of the surface. The affinity values obtained in the analysis are consistent with the numbers required to (a) promote signaling between the correct analyte and the estrogen receptor, and (b) minimize the signaling between xenoestrogens and the estrogen receptor.     

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.     

Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells

We have recently reported that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487-14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (Deltap85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (Deltap85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFkappaB activation through phosphorylation and degradation of IkappaBalpha. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with Deltap85 or the super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFkappaB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and Biophysical properties

Mammalian defensins (alpha as well as beta forms) have a beta-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine beta-defensin-2 (BNBD-2): VRNHVTC(1)RINRGFC(2)VPIRC(3)PGRTRQIGTC(4)FGPRIKC(5)C(6)RSW (positions of disulfide bonds are C(1)[bond]C(5), C(2)[bond]C(4), and C(3)[bond]C(6)). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of beta-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the beta-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.     

Alpha-synuclein association with phosphatidylglycerol probed by lipid spin labels

Alpha-synuclein is a small presynaptic protein, which is linked to the development of Parkinson's disease. Alpha-synuclein partitions between cytosolic and vesicle-bound states, where membrane binding is accompanied by the formation of an amphipathic helix in the N-terminal section of the otherwise unstructured protein. The impact on alpha-synuclein of binding to vesicle-like liposomes has been studied extensively, but far less is known about the impact of alpha-synuclein on the membrane. The interactions of alpha-synuclein with phosphatidylglycerol membranes are studied here by using spin-labeled lipid species and electron spin resonance (ESR) spectroscopy to allow a detailed analysis of the effect on the membrane lipids. Membrane association of alpha-synuclein perturbs the ESR spectra of spin-labeled lipids in bilayers of phosphatidylglycerol but not of phosphatidylcholine. The interaction is inhibited at high ionic strength. The segmental motion is hindered at all positions of spin labeling in the phosphatidylglycerol sn-2 chain, while still preserving the chain flexibility gradient characteristic of fluid phospholipid membranes. Direct motional restriction of the lipid chains, resulting from penetration of the protein into the hydrophobic interior of the membrane, is not observed. Saturation occurs at a protein/lipid ratio corresponding to approximately 36 lipids/protein added. Alpha-synuclein exhibits a selectivity of interaction with different phospholipid spin labels when bound to phosphatidylglycerol membranes in the following order: stearic acid > cardiolipin > phosphatidylcholine > phosphatidylglycerol approximately phosphatidylethanolamine > phosphatidic acid approximately phosphatidylserine > N-acyl phosphatidylethanolamine > diglyceride. Accordingly, membrane-bound alpha-synuclein associates at the interfacial region of the bilayer where it may favor a local concentration of certain phospholipids.     

Platelet-activating factor acetylhydrolase,and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles

Paraoxonase-1 (PON1), an high density lipoprotein (HDL)-associated organophosphate triesterase, suppresses atherosclerosis in an unknown way. Purified PON1 protects lipoprotein particles from oxidative modification and hydrolyzes pro-atherogenic oxidized phospholipids and the inflammatory mediator platelet-activating factor (PAF). We find human PON1 acted as a phospholipase A(2) but not as a phospholipase C or D through cleavage of phosphodiester bonds as expected. PON1 requires divalent cations, but EDTA did not block the phospholipase A(2) activity of PON1. In contrast, a serine esterase inhibitor abolished phospholipase activity even though PON1 has no active-site serine residues. PAF acetylhydrolase, an oxidized phospholipid phospholipase A(2), is a serine esterase associated with specific HDL particles. Western blotting did not reveal detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A(2) activity. We revised the standard PON1 purification by first depleting HDL of PAF acetylhydrolase to find PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF, yet displayed full paraoxonase activity. We conclude that PAF acetylhydrolase is the sole phospholipase A(2) of HDL and that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids.