Cationic Effectors Research Articles

Synergistic regulation of chloride anion recognition using a triple-functional sites receptor with two different cationic effectors.

The synergistic regulation of the multi-functional sites on one receptor molecule with different cationic effectors for anion recognition is scarce to be well understood from the experiment and theory. In this work, a new anion receptor with three functional zones including ether hole, biurea and double bipyridine groups (EUPR) is designed expecting to enhance the chloride anion recognition together with a rational synthesis path being proposed based on four simple and mature organic reaction steps. The conformational structures of the designed receptor EUPR and the binding behaviors for three kinds of ions (Cl-, Na+, and Ag+) are deeply investigated by using density functional theoretical calculations. It is found that Cl- binding via the hydrogen bond interaction can be significantly enhanced and synergistically regulated by the two kinds of cations and the corresponding conformational changes of receptor EUPR. Especially, the conformational pre-organization of receptor caused by the encapsulation of sodium ion into ether hole is benefit to the binding for Cl- in both thermodynamics and kinetics. Na+ binding, in turn, can ever be enhanced by chloride anion, whereas it seems that Ag+ binding cannot always be enhanced by chloride anion, reflecting an electrical complementary matching and mutual enhancement effect for different counter ions. Moreover, solvent effect calculations indicate that EUPR may be an ideal candidate structure for Cl- recognition by strategy of counter ion enhancement in water. Additionally, a visual study of intermolecular noncovalent interaction (NCI) and molecular electrostatic potential (ESP) are used for the analysis on the nature of interactions between receptor and bound ions.

Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation

An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.

Tuning Protein Frameworks via Auxiliary Supramolecular Interactions.

Protein crystals with their precise, periodic array of functional building blocks have potential applications in biomaterials, sensing, and catalysis. This paper describes how a highly porous crystalline framework of a cationic redox protein and an anionic macrocycle can be modulated by a small cationic effector. Ternary composites of protein (∼13 kDa), calix[8]arene (∼1.5 kDa), and effector (∼0.2 kDa) formed distinct crystalline architectures, dependent on the effector concentration and the crystallization technique. A combination of X-ray crystallography and density functional theory (DFT) calculations was used to decipher the framework variations, which appear to be dependent on a calixarene conformation change mediated by the effector. This "switch" calixarene was observed in three states, each of which is associated with a different interaction network. Two structures obtained by co-crystallization with the effector contained an additional protein "pillar", resulting in framework duplication and decreased porosity. These results suggest how protein assembly can be engineered by supramolecular host-guest interactions.

A Unique Family of Neuronal Signaling Proteins Implicated in Oncogenesis and Tumor Suppression.

The neuronal proteins GAP43 (neuromodulin), MARCKS, and BASP1 are highly expressed in the growth cones of nerve cells where they are involved in signal transmission and cytoskeleton organization. Although their primary structures are unrelated, these signaling proteins share several structural properties like fatty acid modification, and the presence of cationic effector domains. GAP43, MARCKS, and BASP1 bind to cell membrane phospholipids, a process reversibly regulated by protein kinase C-phosphorylation or by binding to the calcium sensor calmodulin (CaM). GAP43, MARCKS, and BASP1 are also expressed in non-neuronal cells, where they may have important functions to manage cytoskeleton architecture, and in case of MARCKS and BASP1 to act as cofactors in transcriptional regulation. During neoplastic cell transformation, the proteins reveal differential expression in normal vs. tumor cells, and display intrinsic tumor promoting or tumor suppressive activities. Whereas GAP43 and MARCKS are oncogenic, tumor suppressive functions have been ascribed to BASP1 and in part to MARCKS depending on the cell type. Like MARCKS, the myristoylated BASP1 protein is localized both in the cytoplasm and in the cell nucleus. Nuclear BASP1 participates in gene regulation converting the Wilms tumor transcription factor WT1 from an oncoprotein into a tumor suppressor. The BASP1 gene is downregulated in many human tumor cell lines particularly in those derived from leukemias, which display elevated levels of WT1 and of the major cancer driver MYC. BASP1 specifically inhibits MYC-induced cell transformation in cultured cells. The tumor suppressive functions of BASP1 and MARCKS could be exploited to expand the spectrum of future innovative therapeutic approaches to inhibit growth and viability of susceptible human tumors.

MARCKS as a Negative Regulator of Lipopolysaccharide Signaling

Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.

Allosteric P═O-Based Receptors for Dicarboxylic Acids

The synthesis of new P═O-disubstituted receptors with appended crown ethers and their properties as receptors for dicarboxylic acids have been studied. High affinities have been observed (oxalic and malonic acids with 4-, 5-, 6-, or 8-crown ethers). Binding of a cationic effector within the crown ether unit resulted in a positive "allosteric" effect, which has been determined to be K(rel) = 7 in the best case (binding of malonic acid with Li(+) @ rac-3b).

Cationic composition and acid–base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.

Stepwise and Dramatic Enhancement of Anion Recognition with a Triple-Site Receptor Based on the Calix[4]arene Framework Using Two Different Cationic Effectors

Synthesis and binding behavior of a novel multi-responsive host 1, in which two esters, two polyether moieties, two urea sites, and two bipyridine units as ion binding sites are arranged on the calix[4]arene skeleton, is reported. 1 recognizes Na(+) and Ag(+) simultaneously and quantitatively and captures an anionic guest. The ability of 1 to recognize anions, including CF(3)SO(3)(-) and BF(4)(-), remarkably increases in a stepwise manner using Na(+) and Ag(+) as effectors. The enhancement of the K(a) eventually reaches factors of 1500 and 2000 for NO(3)(-) and CF(3)SO(3)(-), respectively, in the presence of both Na(+) and Ag(+), compared to the free 1. The regulation of binding of multiple ligands may be applicable to multistep cascade systems for the amplification of molecular events, and further studies in this field could provide insight applicable to more advanced molecular devices.

Susceptibility of the guard-cell K(+)-uptake channel KST1 to Zn(2+) requires histidine residues in the S3-S4 linker and in the channel pore

Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K(+) channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K(+)-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn(2+) in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn(2+)-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn(2+), the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn(2+ )sensitivity. Since both histidines alter the susceptibility of KST1 to Zn(2+), the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore.

Activation of the ADP/ATP carrier from mitochondria by cationic effectors

The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low-ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred μM (diaminoalkanes, divalent cations) to 1 μM ( cytochrome c, spermine ) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system.

Photoelectric and spectral responses of bacteriorhodopsin modified by carbodiimide and amine derivatives.

Photoelectric activity and photocycle kinetics of bacteriorhodopsin successively treated with a carbodiimide and several amine derivatives have been studied. Both spectral and electrical responses were initiated by a 15‐ns laser flash inducing a single turnover of bacteriorhodopsin. Photocycle kinetics were measured in a suspension of purple sheets. Photoelectric potential generation was monitored by a voltmeter, using a phospholipid‐impregnated collodion film covered on one side with purple sheets or proteoliposomes.It is shown that in bacteriorhodopsin treated with a carbodiimide derivative and ethylenediamine or methyl‐o‐arginine, significant acceleration of the millisecond phase of the photoelectric response is observed, whereas the kinetics of the microsecond phase remain unchanged. This effect is accompanied by an increase in the fast (τ 2 ms) component of the 412‐nm intermediate decay. Methylamine‐modified bacteriorhodopsin shows no such changes in electric and spectral kinetics.Treatment with ethylenediamine or arginine, rather than methylamine, desensibilizes, completely or partially, photoelectric response to inhibition by cationic effectors such as La3+ or by increase in the H+ concentration. At the same time modification sensibilizes this response to the inhibiting effect of an anion, citrate.Dark adaptation of bacteriorhodopsin modified by ethylenediamine or arginine slows down both microsecond and millisecond electrogeniac phases, whereas that of untreated or methylamine modified bacteriorhodopsin does not.Proteoliposomes of ethylenediamine‐modified bacteriorhodopsin generate a photoelectric potential of opposite direction (interior negative) as compared with those of unmodified or methylamine‐modified bacteriorhodopsin.It is concluded that the modifications by ethylenediamine and arginine change the decomposition of the 412‐nm intermediate in such a fashion that a portion of this intermediate decays faster. This results in accelerating those electrogenic phases which are associated with the 412‐nm intermediate decay. Such an effect may be due to replacement of negatively charged groups in the bacteriorhodopsin polypeptide chain by positively charged ones.Different methods for measurement of changes in the bacteriorhodopsin activity are discussed. The conclusion is made that the analysis of the kinetics of photoelectric potential generation in the purple sheet‐collodion film system is a sensitive probe for changes in bacteriorhodopsin activity.

Studies on a molecular basis for the heparin-induced regulation of enzymatic activity of mouse striatal tyrosine hydroxylase in vitro. Inhibition of heparin activation and of the enzyme by poly-L-lysyltyrosine and poly-L-lysylphenylalanine and their constituent peptides.

Tyrosine hydroxylase was purified up to 10-fold from hypotonic extracts of mouse striatum by heparin affinity chromatography. The purified enzyme (a) had a low Km for tyrosine (around 15 microM) and was not inhibited by tyrosine at concentrations up to 0.2 mM when tetrahydrobiopterin was cofactor and (b) was activated by heparin. The interaction of heparin with tyrosine hydroxylase was studied in ways relating to the known interaction with antithrombin. Heparin and keratan sulfates failed to activate tyrosine hydroxylase in place of heparin; several fractions of the bulk heparin (constituting 5 and 15%) had enriched tyrosine hydroxylase-activating potency; and two lysine copolypeptides ((polylysyltyrosine and polylysylphenylalanine) inhibited the activation of tyrosine hydroxylase by heparin. The lysine copolymers also directly inhibited the enzyme. Heparin (but not heparan and keratan sulfates) protected tyrosine hydroxylase from this inhibition. The constituent lysyltyrosyl (but not lysylphenylalanyl) peptide inhibited tyrosine hydroxylase, and heparin also reversed this inhibition, which was sigmoidal (IC50 of 490 microM) and partially competitive with tyrosine. Tyrosine hydroxylase was purified up to sevenfold by lysyltyrosyl-affinity chromatography. This enzyme preparation exhibited an eightfold greater sensitivity to lysyltyrosylamide than tyrosine hydroxylase purified by heparin affinity. The data indicate that tyrosine hydroxylase is regulated in vitro by a negatively charged site. Occupancy of this site by cationic effectors results in allosteric inhibition which mediates changes in the apparent Km for tyrosine.