Activation and roles of ALK4/ALK7-mediated maternal TGFbeta signals in zebrafish embryo

Activin, Nodal, and Vg1, members of the transforming growth factor beta (TGFbeta) superfamily, transduce signal through type I receptors ALK4 or ALK7 and play important roles in mesoderm induction and patterning during vertebrate embryogenesis. However, the timing and magnitude of the ALK4/ALK7-mediated maternal TGFbeta signals are not clear. SB-431542 is identified as an inhibitor of the ALK4/ALK5/ALK7-mediated TGFbeta signals and its specificity in vertebrate embryos has not been reported. We demonstrate that SB-431542 is able to specifically and reproducibly block the Smad2/3-mediated TGFbeta signals in zebrafish embryo. Embryos exposed to SB-431542 exhibit various defects phenocopying Nodal-deficient mutants. SB-431542 treatments starting at different cell cycles before the midblastula transition lead to different degrees of developmental defects in mesoderm induction and patterning, suggesting that maternal TGFbeta signals are activated right after fertilization and required for mesoderm formation and patterning.     

Structural investigations on the Nodal‐Cripto binding: A theoretical and experimental approach

Nodal, a member of the transforming growth factor‐β superfamily, is a potent embryonic morphogen also implicated in tumor progression. Up to date structural information on the interaction of Nodal with its molecular partners are unknown. To deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto‐dependent tumor progression, we present here a molecular model of activin receptor‐like kinase 4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal, which encompasses residues 43–69 and includes the prehelix loop and the H3 helix. This hypothesis has been subsequently assessed by surface plasmon resonance binding assays between the full‐length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition, the binding affinity between the full‐length Nodal and Cripto proteins has been evaluated for the first time. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1011–1021, 2010.     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

Quantification of helix-helix binding affinities in micelles and lipid bilayers

A theoretical approach for estimating association free energies of alpha-helices in nonpolar media has been developed. The parameters of energy functions have been derived from DeltaDeltaG values of mutants in water-soluble proteins and partitioning of organic solutes between water and nonpolar solvents. The proposed approach was verified successfully against three sets of published data: (1) dissociation constants of alpha-helical oligomers formed by 27 hydrophobic peptides; (2) stabilities of 22 bacteriorhodopsin mutants, and (3) protein-ligand binding affinities in aqueous solution. It has been found that coalescence of helices is driven exclusively by van der Waals interactions and H-bonds, whereas the principal destabilizing contributions are represented by side-chain conformational entropy and transfer energy of atoms from a detergent or lipid to the protein interior. Electrostatic interactions of alpha-helices were relatively weak but important for reproducing the experimental data. Immobilization free energy, which originates from restricting rotational and translational rigid-body movements of molecules during their association, was found to be less than 1 kcal/mole. The energetics of amino acid substitutions in bacteriorhodopsin was complicated by specific binding of lipid and water molecules to cavities created in certain mutants.     

ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left-right axis determination and mesoderm induction in Xenopus

In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.     

Selectivity in heavy metal- binding to peptides and proteins

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.     

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin‐like activities

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI_51–62, and a related analog (P₂) in which a proline residue was inserted in order to mimic the N‐terminal region of the bradykinin molecule. The related retro‐inverso counterparts Ri‐BbKI_51–62 and Ri‐P₂ were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI_51–62 nor its Ri‐BbKI_51–62 analog exhibit bradykinin‐like activity, although the retro‐inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P₂ and Ri‐P₂ presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B‐2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational behavior of fragments of adrenocorticotropin and their antisense peptides determined by NMR spectroscopy and CD spectropolarimetry

An ‘antisense’ peptide (‘HTCA’), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH(1–24) was shown to bind ACTH(1–24) with a K_d of 0.3 nM in a solid-matrix binding assay [(1986) Biochem. J. 234, 679–683]. Two-dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1–24) and ACTH(1–13), as well as their antisense peptides, HTCA and HTCA(12–24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The K_d values are greater than 1 mM.     

Conformational features of a synthetic model of the first extracellular loop of the angiotensin II AT1A receptor.

The angiotensin II AT1A receptor belongs to the G-protein coupled receptors (GPCRs). Like other membrane proteins, GPCRs are not easily amenable to direct structure determination by the currently available methods. The peptide encompassing the putative first extracellular loop of AT1A (residues Thr88-Leu100, el1) has been synthesized along with a cyclic model where the linear peptide has been covalently linked to a template designed to keep the distance between the peptide termini as expected in the receptor. The conformational features of the two molecules have been studied using circular dichroism and NMR techniques. The region W94PFG97 forms a type-II beta-turn and undergoes a Trp-Pro peptide bond cis-trans isomerization in both peptides confirming that these characteristics are intrinsic to el1. In addition, the presence of the spacer seems to modulate the flexibility of the peptide.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

ALK5 and Smad4 are involved in TGF-beta1-induced pulmonary endothelial permeability

The ability of inflammatory cytokine TGF-beta1 to alter endothelial cell phenotype suggests its role in the regulation of vascular endothelial cell permeability. We demonstrate that depletion of TGF-beta1 receptor ALK5 and regulatory protein Smad4, but not ALK1 receptor attenuates TGF-beta1-induced permeability increase and significantly inhibits TGF-beta1-induced EC contraction manifested by actin stress fiber formation and increased MLC and MYPT1 phosphorylation. Consistent with these results, EC treatment with SB 431542, an inhibitor of ALK5 but not ALK1 receptor, significantly attenuates TGF-beta1-induced permeability. Thus, our data demonstrate for the first time direct link between TGF-beta1-mediated activation of ALK5/Smad and EC barrier dysfunction.     

Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Dissection of RAP-LRP interactions: binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP

The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7-CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7-CR8, with the higher affinity site (K(d) approximately 1microM) located in the C-terminal two-thirds and the weaker site (K(d) approximately 5microM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5-CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding.     

Conformational changes in DNA‐binding proteins: Relationships with precomplex features and contributions to specificity and stability

Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA‐binding proteins by superimposing DNA‐bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA‐binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Proteins 2014; 82:841–857. © 2013 Wiley Periodicals, Inc.     

Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia-associated Rho guanine nucleotide exchange factor

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.     

Two neuronal nicotinic acetylcholine receptors, alpha4beta4 and alpha7, show differential agonist binding modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.     

Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Conformational and structural analysis of the equilibrium between single- and double-strand beta-helix of a D,L-alternating oligonorleucine

Alternating sequences of D and L residues in peptides are directly related to the formation of several kinds of regular helical conformations usually called beta-helices. The major feature of these structures is that they can be associated with the transmembrane ion-conducting channel activity in some natural antibacterial peptides. The study of alternating D,L synthetic peptides is critical to understand how factors such as surrounding media, main chain length, type of side chain and terminal groups, among others, can determine the adoption of a specific kind of beta-helix. Early studies pointed out that the peptides Boc-(D-NLeu-L-NLeu)(6)-D-MeNLe-L-Nl-D-Nl-L-Nl-OMe (Boc: tert-butyloxycarbonyl) and Boc-L-Nle-(D-Nle-L-Nle)(5)-D-MeNle-L-Nle-D-Nle-L-Nle-OMe adopt in chloroform a unique detectable conformation single beta(4.4)- and double beta(5.6) upward arrow downward arrow -helix, respectively. The influence of terminal groups on the final stable conformation of N-formylated peptides has been studied in this work. The initial basic NMR data analysis of a synthetic alternating D,L-oligopeptide with ten norleucines, N-methylated on the residue 7 and having HCO- and -OMe as terminal groups clearly indicates the coexistence of two different conformations in equilibrium. NMR data and molecular dynamics calculations point to a dimeric antiparallel beta-helix structure beta(5.6) upward arrow downward arrow for the main conformation. On the other hand, NMR data suggest a single beta-helix structure beta(4.4) for the second conformation. Finally, a thermodynamic analysis of the equilibrium between both conformations has been carried out by one-dimensional NMR measurements at ten different temperatures. The temperature at which 50% of dimer conformation is dissociated is 319 K. In addition, the dimer-monomer equilibrium curve obtained shows a DeltaG>0 for the whole range of studied temperatures, and its behavior can be considered similar to the thermodynamic denaturation protein processes.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.