A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry.

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.     

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.     

Preferred conformations of a linear RGD tripeptide.

Conformational study of RGD tripeptides in the nonhydrated and hydrated states was carried out using an empirical potential function ECEPP/3 and the hydration shell model in order to investigate preferred conformations and factors responsible for their stability. RGD tripeptides in the nonhydrated and hydrated states can be interpreted as existing as an ensemble of feasible conformations rather than as a single dominant conformation from the analysis of distributions of backbone conformations, hydrogen bonds and beta-turns. The different distributions of conformations for the neutral and zwitterionic RGD tripeptides in both states may indicate that the conformation of the RGD tripeptide is liable to depend on solvent polarity and pH values. beta-Turn populations for the neutral tripeptide in both states are reasonably consistent with NMR measurements on linear RGD-containing peptides. The degradation of RGD tripeptide seems to be attributed mainly to the hydrogen bonds between the Asp side-chain and the backbone of Asp residue or C-terminal NHMe group, rather than to the flexible backbones of Gly and Asp residues.     

Effects of amino acid substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on fertilization and parthenogenetic development in mature bovine oocytes

Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al., 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.     

Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.     

Partial atomic charges of amino acids in proteins

Using a semiempirical quantum mechanical procedure (FCPAC) we have calculated the partial atomic charges of amino acids from 494 high-resolution protein structures. To analyze the influence of the protein's environment, we considered each residue under two conditions: either as the center of a tripeptide with PDB structure geometry (free) or as the center of 13-16 amino acid clusters extracted from the PDB structure (buried). The partial atomic charges from residues in helices and in sheets were separated. The FCPAC partial atomic charges of the Cbeta and Calpha of most residues correlate with their helix propensity, positively for Cbeta and negatively for Calpha (r2 = 0.76 and 0.6, respectively). The main consequence of burying residues in proteins is the polarization of the backbone C=O bond, which is more pronounced in helices than in sheets. The average shift of the oxygen partial charges that results from burying is -0.120 in helix and -0.084 in sheet with the charge of the proton as unit. Linear correlations are found between the average NMR chemical shifts and the average FCPAC partial charges of Calpha (r2 = 0.8-0.85), N (r3 = 0.67-0.72), and Cbeta (r2 = 0.62) atoms. Correlations for helix and beta-sheet FCPAC partial charges show parallel regressions, suggesting that the charge variations due to burying in proteins differentiate between the dihedral angle effects and the polarization of backbone atoms.     

The formation and activity of PP2A holoenzymes do not depend on the isoform of the catalytic subunit

The protein phosphatase 2A holoenzyme is composed of one catalytic C subunit, one regulatory/scaffolding A subunit, and one regulatory B subunit. The core enzyme consists of A and C subunits only. The A and C subunits both exist as two closely related isoforms, alpha and beta. The B subunits belong to four weakly related or unrelated families, designated B, B', B", and B"', with multiple members in each family. The existence of two A and two C subunit isoforms permits the formation of four core enzymes, AalphaCalpha, AalphaCbeta, AbetaCalpha, and AbetaCbeta, and each core enzyme could in theory give rise to multiple holoenzymes. Differences between Calpha and Cbeta in expression and subcellular localization during early embryonic development have been reported, which imply that Calpha and Cbeta have different functions. To address the question of whether these differences might be caused by enzymatic differences between Calpha and Cbeta, we purified six holoenzymes composed of AalphaCalpha or AalphaCbeta core enzyme and B subunits from the B, B', or B" families. In addition, we purified four holoenzymes composed of AbetaCalpha or AbetaCbeta and B'alpha1 or B"/PR72. The phosphatase activity of each purified form was assayed using myelin basic protein and histone H1 as substrates. We found that Calpha and Cbeta have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Calpha and Cbeta in binding A or B subunits. These data suggest that the distinct functions of Calpha and Cbeta are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Calpha and Cbeta are discussed.     

Structure and function of RGD peptides derived from disintegrin proteins

The Arg-Gly-Asp (RGD) sequence serves as the primary recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the biological activities of matrix proteins. We have initiated structure-function studies of two RGD containing peptides, RGD-5(AGGDD) and cyclic RGD-6(CARGDDC). Assays have shown that cyclic RGD-peptides inhibit platelet aggregation more efficiently than linear ones. NMR data revealed that RGD-5 and RGD-6 have entirely different conformation. RGD-5 has a linear extended structure and RGD-6 has a stable loop conformation. In RGD-5 the guanidinium group of Arg2 and the carboxyl group of Asp4 lie in parallel, whereas the side-chains of Arg3 and Asp5 of RGD-6 are located in different planes, supporting the idea that the stability of the cyclic form derives from the packing of the side chain of the Arg and Asp residues. The structural features of these peptides could provide a basis for designing new drugs against diseases related to platelet aggregation and as cancer antagonists.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.     

Structural studies of a neuropeptide precursor protein with an RGD proteolytic site

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.     

Escherichia coli ilvN interacts with the FAD binding domain of ilvB and activates the AHAS I enzyme

The unique multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been exploited to generate polypeptide fragments which, when cloned and expressed, reassemble in the presence of cofactors to yield a catalytically competent enzyme. Multidimensional multinuclear NMR methods have been employed for obtaining near complete sequence specific NMR assignments for backbone HN, 15N, 13Calpha and 13Cbeta atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in 2H, 13C and 15N. Unambiguous assignments were obtained for 169 of 177 backbone Calpha atoms and 127 of 164 side chain Cbeta atoms. The secondary structure determined on the basis of observed 13Calpha secondary chemical shifts and sequential NOEs agrees well with the structure of this domain in the catalytic subunit of yeast AHAS. Binding of ilvN to the ilvBalpha and ilvBbeta domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvBalpha and ilvBbeta domains of the catalytic subunit and not with the ilvBgamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBbeta and proximal to the intrasubunit ilvBalpha/ilvBbeta domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Designed synthetic analogs of the α‐helical peptide temporin‐La with improved antitumor efficacies via charge modification and incorporation of the integrin αvβ3 homing domain

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin‐La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin‐La analog (temporin‐Las) and related constructs containing the Arg‐Gly‐Asp (RGD) tripeptide, the integrin αvβ3 homing domain (RGD‐La and RGD‐Las). We detected a link between the net charges and integrin αvβ3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin‐La and its synthetic analogs inhibited cancer cell proliferation in a dose‐dependent manner. Evidence was provided that the affinity between RGD‐Las and tumor cell membranes was stronger than other tested peptides using a pull‐down assay. Morphological changes on the cell membrane induced by temporin‐La and RDG‐Las, respectively, were examined by scanning electron microscopy. Additionally, time‐dependent morphological changes were detected by confocal microscopy, where the binding process of RGD‐Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD‐Las. These AMP‐derived peptides have clinical potential as specifically targeting agents for the treatment of αvβ3 positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework

Summary Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motifcontaining sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chain and connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6–11 disulfide bond within the A chain. The two RGD-containing insulin genes were over-expressed inE. coli, and purified and designated as RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those of expected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3 μM, while the latter was 3.5-fold less active. Thein vivo assay also indicated that the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.     

Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel,KAT1

Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated. In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane. A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen. These results indicate that Asp(95) assists in the membrane insertion of S3-S4 and that Asp(105) helps in preventing S4 from being releasing into the ER lumen. The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen. A series of constructs containing different C-terminal truncations of S4 showed that Arg(174) was required for correct integration of S3 and S4 into the membrane. Interactions between Asp(105) and Arg(171) and between negative residues in S2 or S3 and Arg(174) may be formed transiently during membrane integration. These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane.     

Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework

Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motif-containing sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS, were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chainand connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6-11 disulfide bondwithin the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designatedas RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those ofexpected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3 M, while the latter was3.5-fold less active. The in vivo assay also indicatedthat the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity.