Biomedical subjects

R W Roeske

Publications and source records attributed to R W Roeske.

At least 19 recordsLinked to original sources

PubMed2001

Artificial ion channels formed by a synthetic cyclic peptide.

A new cyclic peptide 1 having an (LLLD)3 configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self-assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1, cyclo[-Trp-Dap-Leu-D-Ala-Trp-Ser-Val-D-Ala-Trp-Ser-Ile-Gly-] (Dap: L-diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the beta-amino group of Dap may play a role in the conductance of the peptide channels. Fourier-transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of beta-structure analogous to that in the gramicidin A dimer and distinct from the H-bonding pattern of the beta-barrels.

Circular Dichroism↗

PubMed2001

Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation.

Mammalian gonadotropin-releasing hormone (GnRH) receptors preferentially bind mammalian GnRH, which has Arg in position eight. The Glu(7.32(301)) residue, which determines selectivity of the mouse GnRH receptor for Arg(8)-containing GnRH, is Asp(7.32(302)) in the human GnRH receptor. We have confirmed that Asp(7.32(302)) confers selectivity of the human GnRH receptor for Arg(8) of GnRH and investigated the mechanism of this specificity using site-directed mutagenesis and ligand modification. We find that although Arg(8) and Asp(7.32(302)) are required for high-affinity binding of GnRH, conformationally constrained peptides, with D-amino acid substitutions in position six or with a 6,7 gamma-lactam, bind the human GnRH receptor with high affinity, which is independent of the presence of Asp(7.32(302)) in the receptor or Arg(8) in the ligand. The ability of the ligand constraints to compensate for the absence of both Arg(8) and Asp(7.32(302)) indicates that these residues both have roles in stabilizing a high affinity ligand conformation and that their roles are complementary. This suggests that the Arg(8) and Asp(7.32(302)) side chains interact to induce a high affinity conformation of native GnRH. Thus, Asp(7.32(302)) of the human GnRH receptor determines selectivity for mammalian GnRH by its ability to induce a high affinity conformation of its native ligand. However, this initial interaction seems not to contribute to the final ligand-receptor complex. We propose that Arg(8) interacts transiently with Asp(7.32(302)) to induce a high-affinity ligand conformation of GnRH, which then interacts with a binding pocket that is common for both constrained and unconstrained analogs of GnRH.

Amino Acid Substitution↗

PubMed2000

Selection of peptide ligands that bind to acid fibroblast growth factor.

Acid fibroblast growth factor (aFGF) binds to its cell-surface receptors in a heparin-dependent manner. In an attempt to define the aFGF recognition site on fibroblast growth factor receptor 1 (FGFR1), we developed a screening strategy for identifying FGF ligands that bind to the receptor-binding region of FGF. To retain the natural conformation of aFGF during screening, we used biotinylated heparin to immobilize aFGF on a streptavidin-coated dish. A 15-mer phage display peptide library was then screened in the dish and a group of related peptide sequences was identified. These peptide sequences contain two conserved motifs, SSG and VPS, corresponding to two protein sequences of the immunoglobulin-like (Ig-like) domain II of FGFR1 at amino acids 180-182 and 221-223 (CPSSG-VPSDKGNYTC). Further experiments demonstrate that the phage displaying these sequences can specifically bind to aFGF and that the synthesized peptide corresponding in sequence can inhibit mitogenic activity of aFGF. These sequences may thus constitute part of the aFGF-binding region on FGFR1, and the synthesized peptide has the potential to become a therapeutic agent as an aFGF antagonist.

3T3 Cells↗

PubMed1995

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors.

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC50) induced by GnRH at its half-maximal concentration (EC50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC50s varied between half and twice ((1.22-6.06) x 10(-10) M) the GnRH EC50 (3 x 10(-10) M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC50s varying from 46 to 1490 times ((1.4-44.7) x 10(-7) M) the GnRH EC50 (3 x 10(-9) M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.

Animals↗

PubMed1994

Effects of C alpha-methyl substitution on the conformation of linear GnRH antagonist analogs.

We have examined the effect of C alpha-methyl groups on the conformational ensemble of GnRH analog peptides by comparing 1H 2D NMR data from two analogs, Ac-D-Nal1-D-4-Cl-C alpha-Me-Phe2-D-Pal3-Ser4-Tyr5-D-Arg6-Leu7-Arg8-Pro9-D-Al a10- NH2 (1) and Ac-D-Nal1-D-4-Cl-C alpha-Me-Phe2-D-Pal3-Ser4-C alpha-Me-Tyr5-D-Arg6- Leu7-C alpha-Me-Arg8-Pro9-D-Ala10-NH2 (2). The two additional C alpha-methyl groups in residues 5 and 8 of 2 do not influence significantly the pattern of the observable main chain NOE intensities, or of the backbone HN proton chemical shifts, which indicates that they do not produce global changes in the conformational ensemble of the peptide. A local change induced by the substitution was observed in the conformation at D-Arg8-Pro9.

Amino Acid Sequence↗

PubMed1993

Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain.

GTP-binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab-related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca(2+)-dependent or Ca(2+)-independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL (30-47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca(2+)-stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 microM peptide. The peptide (100 microM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence-specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.

Amino Acid Sequence↗

PubMed1993

A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides.

Two continuous assays for protein tyrosine phosphatases (PTPases) have been developed using phosphotyrosine containing peptide substrates. These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at 282 nm or the fluorescence at 305 nm of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation. The procedure is convenient and efficient, since both kcat and Km values can be obtained in a single run. The difference absorption coefficient (delta epsilon) at 282 nm is relatively insensitive to the pH of the reaction media. These techniques were applied to two homogeneous recombinant PTPases employing six phosphotyrosine-containing peptides. Km and kcat values obtained from the progress curve analysis were similar to those determined by the traditional initial rate inorganic phosphate assay. The peptides corresponding to autophosphorylation sites in Neu, p56lck, and p60src proteins show distinct behavior with the Yersinia PTPase, Yop51*, and the mammalian PTPase (PTP1U323). In both cases, the kcat values were relatively constant for all the peptides tested whereas the Km values were very sensitive to the amino acid sequence surrounding the tyrosine residue, especially in the case of Yop51*. Thus, both Yop51* and PTP1U323 show differential recognition of the phosphotyrosyl residues in the context of distinct primary structure of peptide substrates.

Amino Acid Sequence↗

PubMed1993

Amino- and guanidinoacylryanodines: basic ryanodine esters with enhanced affinity for the sarcoplasmic reticulum Ca(2+)-release channel.

Amino- and guanidinoacyl esters of ryanodine were prepared to evaluate the effect of basicity on the binding affinity of these derivatives for the sarcoplasmic reticulum Ca(2+)-release channel (SR CRC). In the presence of DCC and DMAP Cbz-beta-alanine reacts with ryanodine in CH2Cl2 to give O10eq-Cbz-beta-alanylryanodine (3a), which on hydrogenolysis yields the beta-alanyl ester (4a). N,N'-bis-Cbz-S-methylthiourea reacts with 4a to yield beta-N,N'-bis-Cbz-guanidinopropionylryanodine (5a). O10eq-beta-guanidinopropionylryanodine (6a) is obtained on hydrogenolytic deprotection of 5a. The binding affinity of beta-alanine ester (4a) and its glycyl congener (4b) is 2-3-fold greater, and that of the beta-guanidinopropionyl ester (6a) and its acetyl congener (6b) 3-6-fold greater, than that of ryanodine. The effect of ryanodine on SR Ca2+ flux is of a biphasic nature: nanomolar levels open (activate) the channel, while micromolar levels close (deactivate) it. The base-substituted esters 4a and 6a both display a unidirectional effect: they only open the channel. An understanding of ryanodine's mode of action and the design of effective SR CRC activating and deactivating ryanoids for possible therapeutic application are major research objectives.

Amino Acids↗

PubMed1993

Synthesis of phosphotyrosine-containing peptides by the solid-phase method. A re-examination of the use of Boc-Tyr(PO3Bzl2)-OH.

A practical and convenient procedure for making phosphotyrosine-containing peptides by the solid-phase method was developed. Phosphotyrosine was incorporated via Boc-Tyr(PO3Bzl2)-OH. The completed peptide was cleaved from the solid support by treatment with 1 M TMSBr-thioanisole-TFA. By gel-phase 31P-NMR spectroscopy we found that one of the benzyl protecting groups on phosphate was completely removed by two consecutive runs of Boc deprotection with 50% TFA-DCM. However, the other benzyl group remained intact throughout the synthesis (35 cycles).

Amino Acid Sequence↗

PubMed1993

Effects of thiol protease inhibitors on cell cycle and proliferation of vascular smooth muscle cells in culture.

Smooth muscle proliferation is a prominent feature of the vascular response to mechanical injury. Accordingly, modulation of proliferation has important therapeutic implications for angioplasty restenosis. We have identified a subclass of thiol protease inhibitors (TPIs) that reversibly inhibit bovine aortic smooth muscle cell (BASMC) proliferation in vitro. To define the nature of this inhibition, an evaluation of selected steps in the cell cycle was undertaken. Treatment of BASMCs with benzyloxycarbonyl-Leu-norleucinal (calpeptin) at 100 microM and acetyl-Leu-Leu-norleucinal (TPI-1) at 50 microM was shown to cause a block of platelet-derived growth factor-BB as well as serum-inducible cell cycle progression at a point before the G1-S boundary, reducing the percentage of bromodeoxyuridine-positive cells from 87% to 5% over a 24-hour labeling period. Addition of TPI-1 at various times after serum addition to serum-deprived BASMCs showed 80% of the maximal block of DNA synthesis even when added 6 hours after serum. The cell cycle progression block was gradually lost as the delay from serum to TPI-1 application was increased from 6 to 12 hours. By Northern analysis of mRNA after serum addition, TPI-1 caused a fourfold decrease in the transient elevation of fos and myc proto-oncogene as well as a decrease in the levels of both muscle and nonmuscle actin mRNA induced early after serum addition. Flow cytometric analysis of DNA content and synthesis in BASMCs treated with TPI-1 or calpeptin additionally revealed the presence of a distinct cell cycle block in the G2-M compartment. In the aggregate, these results suggest the existence of more than one molecular site potentially involved in inhibition by TPI of cell cycling in BASMCs.

Animals↗

PubMed1992

Synthesis of optically pure C alpha-methyl-arginine.

Optically pure L-(+)-C alpha-methyl-arginine and D-(-)-C alpha-methyl-arginine were synthesized. Experimental results indicated that DL-C alpha-methyl-arginine methyl ester could be resolved by trypsin, but workup posed a technical difficulty. Chemical resolution at the stage of DL-C alpha-methyl-ornithine, followed by selective guanidination using N,N'-di-Cbz-S-methylisothiourea and hydrogenolysis provided a effective and practical method for the synthesis of optically pure C alpha-methyl-arginine.

Arginine↗

PubMed1991

Guanidination of a peptide side chain amino group on a solid support.

A novel guanidination method of converting a peptide side chain amino group to a guanidino group on a solid support is described. Four guanidinating reagents were evaluated using a model tetrapeptide attached to a polystyrene resin. Experimental data indicate that the two nitroguanidinating reagents, but not the two tosylguanidinating reagents, can be used effectively in solid phase peptide synthesis.

Benzhydryl Compounds↗

PubMed1990

Phosphate groups as substrate determinants for casein kinase I action.

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.

Amino Acid Sequence↗

PubMed1990

Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates.

Recognition of substrates by the protein kinase glycogen synthase kinase 3 (GSK-3) usually requires prior phosphorylation of the substrate. Using a peptide based on the glycogen synthase sequence PRPAS(3a)VPPS (3b)PSLS(3c)RHSS(4)PHQS(5)EDEEEP (where the numbers in parentheses denote sites of phosphorylation), we showed previously that phosphorylation of site 5 by casein kinase II was necessary for GSK-3 to phosphorylate the peptide at sites 3a, 3b, 3c, and 4 (Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048). In the present study, variant peptides were synthesized in which sites 3a, 3b, 3c, and 4 were individually replaced by Ala residues (denoted Ala-3c, etc.). All of the variant peptides were substrates for casein kinase II. The peptide Ala-4,Ser(P)-5 was not a substrate for GSK-3 confirming the minimal recognition sequence for the protein kinase as -SXXXS(P)-. The peptides Ala-3c,Ser(P)-5, Ala-3b,Ser(P)-5, and Ala-3a,Ser(P)-5, however, were all good substrates for GSK-3 with apparent Km values in the range 3-6 microns, comparable with that of the parent peptide. GSK-3 could introduce 1, 2, and 3 phosphates, respectively, into these substrates, always COOH-terminal to the substituted Ala residue. Ala-4,Ser(P)-5 and Ala-3c,Ser(P)-4,Ser(P)-5 were competitive inhibitors for phosphorylation of the parent peptide, with Ki values of 2 and 5 microns, respectively. The data suggest (i) that GSK-3 recognizes serines in the motif -SXXXS(P)-, and (ii) that multiple phosphorylation of the peptide substrate has an obligate order, with the sequential formation of new recognition sequences.

Amino Acid Sequence↗

PubMed1989

Synthesis and channel properties of [Tau 16]gramicidin A.

Des(ethanolamine)-taurine16-gramicidin A ([Tau 16]gramicidin A) was synthesized by the solid phase method and its channel-forming behavior in planar lipid bilayers was examined. The purified monovalent anionic peptide formed channels when applied to the aqueous compartments on both sides of the bilayer, but not when applied to one side only. The single-channel conductance was measured for KCl concentrations between 0.1 and 1.0 M and was found to be higher than that of gramicidin A in each case. Single-channel lifetimes were similar to those of gramicidin A suggesting that the channels have the beta 6.3 helix structure.

Gramicidin↗

PubMed1988

Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1.

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.

Arginine↗

PubMed1987

Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3.

The mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.

Animals↗

PubMed1987

Cell surface specific immunoglobulin inhibits alpha factor mediated morphogenesis in Saccharomyces cerevisiae.

Immunoglobulins raised from Saccharomyces cerevisiae a and alpha mating type cell envelope preparations inhibited alpha factor mediated morphogenesis of the a cell without inhibiting normal cell division. The Ig responsible for this inhibition was absorbed to both a and alpha whole cells and heat-killed cells, indicating that the immunoglobulin binding sites were exposed on the cell surface and not mating type specific. Additionally, alpha factor mediated cell cycle arrest was not affected by the immunoglobulin preparations, implying that the immunoglobulin was not preventing alpha factor from binding to its receptor.

Antigens, Surface↗