Cytomodulin-1, a synthetic peptide abrogates oncogenic signaling pathways to impede invasion and angiogenesis in the hamster cheek pouch carcinogenesis model.

Constitutive activation of the various oncogenic signaling pathways plays a pivotal role in promoting malignant transformation. The aim of this study was to investigate the therapeutic potential of a synthetic bioactive heptapeptide cytomodulin-1 (CM-1) against hamster cheek pouch carcinomas based on its influence on the predominant carcinogenic signaling pathways - NF-κB, TGFß, and Wnt/ß-catenin and their downstream target events invasion and angiogenesis. Topical application of CM-1 to DMBA-painted hamsters significantly inhibited activation of the canonical NF-κB pathway by blocking kinase activity of IKKß and increasing the cytosolic accumulation of the inhibitor IκB-α. In addition, CM-1 inactivated IKKß by disrupting IKKß/Nemo interactions. CM-1 also hampered the activation of TGFß and Wnt/ß-catenin signaling by averting the phosphorylation of the key upstream ser/thr kinases TGFß RI and GSK-3ß respectively. Attenuation of these oncogenic signaling pathways by CM-1 also mitigated invasion and angiogenesis by suppressing the expression of pro-invasive matrix metalloproteinases, pro-angiogenic VEGF and HIF-1α and upregulating the anti-angiogenic TIMP-2. Synthetic peptides such as CM-1 that target multiple key molecules in oncogenic signaling pathways and their downstream events are ideal candidate agents for cancer chemotherapy.

Synthetic peptides cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2) promote collagen synthesis and wound healing in vitro.

Transforming growth factor-beta1 (TGF-beta1) is believed to be a key player in wound healing, promoting cell proliferation, migration, and matrix synthesis in a variety of cell types. We have designed two peptides, i.e., cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2), to simulate the binding domain of TGF-beta1. In this study we examined the bioactivity of the two synthetic peptides CM-1 and CM-2 on human foreskin fibroblasts (HFF). Synthetic peptides CM-1 and CM-2 in culture media increased wound healing of fibroblasts in an injury model in vitro. In addition, CM-1 and CM-2 enhanced the gene expression of collagen I and increased the production of pro-collagen I peptide in HFF. These results suggest that CM-1 and CM-2 have potentially useful clinical applications in wound healing.

Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype.

Cartilage formation during embryonic development and in fracture healing in adult animals involves chondrogenic differentiation of mesenchymal precursors. Here we describe an in vitro model whereby human dermal fibroblasts, considered to be restricted to a fibroblast lineage, are apparently redirected toward a chondrogenic phenotype by high density micromass culture in the presence of lactic acid. Micromass cultures treated with 40 mM lactate exhibited increased levels of Alcian blue staining and sulfate incorporation, indicative of elevated sulfated glycosaminoglycan synthesis. Northern analysis revealed an up-regulation of chondroitin sulfate proteoglycan 1 (aggrecan) and transforming growth factor-beta 1 mRNA and a decrease in type I collagen expression. Type II collagen was detected by reverse transcription-PCR only in experimental cultures. Although the observed changes in biosynthesis and gene expression were consistent with differentiating chondrocytes, the cells displayed an elongated, fibroblast-like morphology. These findings suggest that dermal fibroblasts may be committed to differentiate along a chondrogenic pathway by in vitro culture under specific forcing conditions.

The structure of myristoyl-CoA:protein N-myristoyltransferase.

Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes. Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt). Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs. The enzyme is also a potential target for development of antiviral and antineoplastic agents. The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs. The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily. The structure reveals how Nmt's myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzyme's ordered kinetic mechanism. Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmt's C-terminal carboxylate. The use of main chain carboxylate atoms as general base catalyst is a novel feature.

The effects of colchicine on a Peyronie's-like condition in an animal model.

PURPOSE: We have developed an animal model of Peyronie's disease by injecting transforming growth factor beta (TGF-beta) into the rat penis. Our objective is to study the effects of colchicine on the Peyronie's condition in an animal model. MATERIALS AND METHODS: Thirty-six adult male Sprague-Dawley rats received TGF-beta injections into the tunica albuginea and were divided into two groups (n = 18 each). Rats in the first group were divided into three subgroups (n = 6 each). Each rat in the three subgroups received the following: Subgroup 1 received colchicine, subgroup 2 received ibuprofen, and subgroup 3 received regular water. The rats were euthanized after 6 weeks. Rats in the second group were also divided into three subgroups. These rats received the same treatments as the rats in the first group, but treatments began 6 weeks after TGF-beta injection. These rats were euthanized after 12 weeks. Tunical tissue samples were collected and examined using Hart and trichrome stains, electron microscopy (EM), and western blot analysis for TGF-beta detection. RESULTS: In the first group, the colchicine-treated rats exhibited less collagen deposition and less elastic fiber fragmentation than the untreated or ibuprofen-treated rats. EM confirmed the results and showed normal distribution and shape of both collagen and elastic fibers in the colchicine-treated group. In the second group, the colchicine-treated rats exhibited less crowding of the collagen fibers. However, the elastic fibers remained fragmented and scarce. Western blot analysis showed significant down-regulation of TGF-beta expression (5/6) in the colchicine-treated group after 6 weeks. Down-regulation was observed in only 1/6 in both ibuprofen and non-treated groups. After 12 weeks 2/6, 1/6, and 1/6 rats displayed down regulation in the colchicine treated, ibuprofen treated, and non-treated groups, respectively. CONCLUSION: Early colchicine treatment may suppress a Peyronie's like condition in the rat animal model.

Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.

In tissues, collagen forms the scaffold for cell attachment and migration, and it modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli. We are constructing biomimetic environments by immobilizing a collagen-derived high-affinity cell-binding peptide P-15 in three-dimensional (3-D) templates. The cell-binding peptide can be expected to transduce mechanical forces. In their physiological environment, periodontal ligament fibroblasts (PDLF) are subject to significant mechanical forces. We have examined the behavior of human PDLF in culture on particulate bovine anorganic bone mineral (ABM) coated with P-15 (ABM-P-15). Greater numbers of cells associated with ABM-P-15 compared to ABM alone. Higher levels of incorporation of radiolabeled precursors in DNA and protein were consistent with the presence of larger numbers of cells on ABM-P-15 compared to ABM cultures. Scanning electron microscopic examination showed that cultures on ABM-P-15 generated highly oriented 3-D colonies of elongated cells and formed copious amounts of fibrous as well as membranous matrix reminiscent of ligamentous structures. PDLF cultured on ABM formed sparse monolayers with little order and a meager matrix. Alizarin Red stained the matrix of particle associated cells and inter-particle cellular bridges in P-15-associated cultures, indicating mineralization. 3-D colony formation and ordering of cells along with increased mineralization suggests that the coupling of cells to the ABM matrix through P-15 may provide a biomimetic environment permissive for cell differentiation and morphogenesis. Our studies suggest that ABM-P-15 templates may be effective as endosseous grafts, and, when seeded with PDLF, these matrices may serve as tissue engineered substitutes for autologous bone grafts.

Differential stability of the triple helix of (Pro-Pro-Gly)10 in H2O and D2O: thermodynamic and structural explanations.

(Pro-Pro-Gly)10 [(PPG10)], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5 degrees C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D2O to an H2O solution of (PPG)10, with the melting temperature reaching 40 degrees C in pure D2O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalpy of unfolding in D2O vs. H2O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)10 that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H2O or D2O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D2O resulted in a significant lowering of the potential energy of hydrated (PPG)10. This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG10) energy, plus the water-(PPG10) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D2O vs. H2O. This result indicates that the geometry of the carbonyl-D2O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H2O.

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs.

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 A resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzyme's substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzyme's C-terminal backbone carboxylate.

The role of bound water in the stability of the triple-helical conformation of (Pro-Pro-Gly)10.

There is significant experimental evidence for bound water in collagen and related polymers. (Pro-Pro-Gly)10 [(PPG)10] is a polymer that forms a collagen-like triple-helical structure in aqueous solution. Like collagen, (PPG)10 adopts a structure in which side chains are mostly exposed to solvent, and the backbone polar groups are limited in their ability to form hydrogen bonds with each other. (PPG)10, like collagen, also has many of its backbone polar groups in positions that inhibit complete solvation in aqueous solution; thus the necessity of bound waters for stabilization of the structure. We have constructed a model for bound waters in (PPG)10, based on an examination of the geometry and steric environment of the backbone polar groups. As will become clear, the number of bound waters is determined by the geometry of the backbone carbonyl groups and the steric crowding surrounding them. In this model, each water forms one hydrogen bond with each of two backbone carbonyls from a glycine and a proline in different monomer chains, thus bridging the two carbonyls. The carbonyls in question are quite sterically crowded by neighboring (PPG)10 atoms and would not be likely to experience complete solvation by bulk solvent in aqueous solution. The bound waters are therefore likely to be present even in solution, since otherwise the unsatisfied hydrogen-bonding potential of the carbonyls would destabilize the structure. Other carbonyls also are sterically crowded and possibly prevented from experiencing full solvation, but are not in a favorable geometry for such bridging hydrogen bonds. The intra- and interchain interactions found in a previous computational study of (PPG)10 without bound waters are not disrupted by the addition of waters.

Histological and ultrastructural alterations in an animal model of Peyronie's disease.

OBJECTIVE: To determine the role of transforming growth factor beta (TGF-beta), one of the cytokines known to induce tissue fibrosis, in the induction of a Peyronie's-like condition, and to produce an animal model for the further study of Peyronie's disease. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were divided into two groups: in group 1, different concentrations of cytomodulin, a synthetic heptapeptide with TGF-beta-like activity, were injected into the tunica of each of 18 rats and six rats group 2 received saline injections as a control. The tunical tissues were taken after 3 days, 2 and 6 weeks and were examined histologically using Hart and trichrome stains. Electron microscopy was used to examine the ultrastructural changes in the same tissue samples. RESULTS: There were histological and ultrastructural alterations in 15 of 18 rats in group 1 (cytomodulin-injected), especially in tissue examined after 6 weeks. The most prominent histological changes were chronic inflammatory cellular infiltration, focal and diffuse elastosis, thickening, disorganization and clumping of the collagen bundles. The ultrastructural changes were in the form of densely packed collagen, fragmented and scarce elastic fibres, separation of neuronal fibres by interposing clumps of packed collagen, and perivascular collagen deposition as a part of the reorganization of the interstitial matrix. CONCLUSION: Cytomodulin can induce a Peyronie's-like condition in the rat penis, which may explain the role of TGF-beta in the pathogenesis of Peyronie's disease. With further refinement, such rats may be used as an experimental model for studies of Peyronie's disease.

Fibroblasts derived from tissue explants of dilantin-induced gingival hyperplasia and idiopathic gingival fibromatosis show distinct disparity in proliferative responsiveness to epidermal growth factor.

Human gingival fibroblasts derived from tissue explants of two patients with dilantin-induced gingival hyperplasia (DGH) and one patient with idiopathic gingival fibromatosis (GF) were studied with respect to the effect of epidermal growth factor (EGF) on the proliferative characteristics of these cells. Immunohistochemical staining showed that there were more EGF receptor-positive cells among DGH fibroblasts than among either normal gingival fibroblasts (NG) or GF cells. Furthermore, EGF binding studies showed that, in spite of there being no disparity in binding affinity among all these cells, DGH fibroblasts possessed approximately two-fold more EGF receptors than either NG or GF cells. In addition, the growth-promoting effect of exogenously added EGF was concentration-dependent in DGH fibroblasts but was not in either NG or GF cells. All of the above findings clearly demonstrate that DGH and GF fibroblasts exhibit distinct disparity in proliferative responsiveness to EGF and suggest that different mechanisms may be involved in the pathogenesis of these two forms of gingival hyperplasia. These observations also suggest a possible therapeutic approach for blocking EGF-induced cell proliferation in DGH.

An animal model of Peyronie's-like condition associated with an increase of transforming growth factor beta mRNA and protein expression.

PURPOSE: Transforming growth factor beta (TGF-beta) is involved in numerous vital processes including tissue fibrosis. Our objective was to study the role of TGF-beta in the induction of a Peyronie's-like condition and to produce an animal model for the further study of Peyronie's disease. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were divided into two groups. Different concentrations of cytomodulin, a synthetic heptopeptide with TGF-beta-like activity, were injected into the tunica of each rat from the first group (n = 18). Rats in the second group (n = 6) received saline injections as a control. The tunical tissues were taken after 3 days, 2 weeks, and 6 weeks and were examined using Hart and Trichrome stains. In the same tissue samples, TGF-beta mRNA and protein expression were studied. RESULTS: Histological alterations were observed in 15 out of 18 cytomodulin-injected rats, especially in tissue examined after 6 weeks. The most prominent changes were chronic cellular infiltration, focal and diffuse elastosis, thickening, disorganization and clumping of the collagen bundles. Results from immunoblot revealed remarkable TGF-beta1 protein expression in all the cytomodulin-injected rats only after 2 and 6 weeks. No remarkable TGF-beta2 or TGF-beta3 protein expression was observed. TGF-beta1 mRNA expression in the cytomodulin-injected rats was noticed in rats injected with higher concentrations after 3 days, while it was expressed in all rats after 2 weeks. There was no expression in the control group after either 3 days or 2 weeks. CONCLUSIONS: Cytomodulin can induce Peyronie's-like condition in the rat penis, which may explain the role of TGF-beta in the pathogenesis of Peyronie's disease.

Titration calorimetric analysis of AcylCoA recognition by myristoylCoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3'-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a Kd of 15 nM and a large exothermic deltaH (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller deltaH and a correspondingly less negative TdeltaS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3'-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3'-phosphate have distinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.

The role in cell binding of a beta-bend within the triple helical region in collagen alpha 1 (I) chain: structural and biological evidence for conformational tautomerism on fiber surface.

In its physiological solid state, type I collagen serves as a host for many types of cells. Only the molecules on fiber surface are available for interaction. In this interfacial environment, the conformation of a cell binding domain can be expected to fluctuate between the collagen fold and a distinctive non-collagen molecular marker for recognition and allosteric binding. If the cell binding domain can be localized in contiguous residues within the exposed half of a turn of the triple helix (approximately 15 residues), the need for extensive structural modification and unraveling of the triple helix is avoided. We examined the conformational preferences and biological activity of a synthetic 15-residue peptide (P-15), analogous to the sequence 766GTPGPQGIAGQRGVV780 in the alpha 1 (I) chain. Theoretical studies showed a high potential for a stable beta-bend for the central GIAG sequence. The flanking sequences showed facile transition to extended conformations. Circular dichroism of the synthetic peptide in anisotropic solvents confirmed the presence of beta-strand and beta-bend structures. P-15 inhibited fibroblast binding to collagen in a concentration dependent manner, with near maximal inhibition occurring at a concentration of 7.2 x 10(-6) M. The temporal pattern of cell attachment was altered markedly in the presence of P-15. No inhibition was seen with a peptide P-15(AI), an analogue of P-15 with the central IA residues reversed to AI or with collagen-related peptides (Pro-Pro-Gly)10, (Pro-Hyp-Gly)10, and polyproline, and with several unrelated peptides. Our studies suggest a molecular mechanism for cell binding to collagen fibers based on a conformational transition in collagen molecules on the fiber surface. Since the energy barrier between the collagen fold and beta-strand conformation is low, a local conformational change may be possible in molecules on the fiber surface because of their location in an anisotropic environment. Our observations also suggest that the sequence incorporated in P-15 may be a specific ligand for cells. Unlike other reported cell binding peptides, the residues involved in this interaction are non-polar.

Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle.

Much effort has been expended on the in vitro characterization of enzymes that covalently attach lipids to proteins. Less information is available about properties conferred on modified proteins by their attached lipid groups, but biophysical studies of simple model systems have begun to shed light on this issue. Recent evidence suggests that the specificity of lipid modifications may be dependent upon the intracellular compartmentalization of the lipid and protein substrates of lipidating enzymes. The function and targeting of their lipidated products appear to be regulated dynamically through addition or subtraction of lipid moieties, other covalent or noncovalent modifications, as well as several devices that at this point can only be inferred. This field of research illustrates the necessity of integrating cell-biological and biophysical perspectives.

Enhanced cell attachment to anorganic bone mineral in the presence of a synthetic peptide related to collagen.

We have examined the ability of a synthetic 15-residue peptide, P-15, related to a biologically active domain of type I collagen, to promote attachment of human dermal fibroblasts to anorganic bovine bone mineral (ABM) phase. The attachment of cells increased with increasing content of P-15 on the surface of ABM particles, as seen by the increased binding of radiolabeled cells, and by light microscopy and scanning electron microscopy. Incorporation of radioactive precursors of DNA and protein synthesis showed that cells on P-15-coated ABM synthesized over twofold the amount of DNA and protein than did cells on uncoated ABM. Fibroblasts attached to ABM in the presence of P-15 formed three-dimensional colonies. Cellular bridges formed between adjacent particles which aggregated in clusters with tissue-like structure. Cultures on ABM.P-15 stained for alkaline phosphatase. These observations suggest that P-15-coated ABM may be a useful matrix for bone repair.

Isothermal titration calorimetric studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. Determinants of binding energy and catalytic discrimination among acyl-CoA and peptide ligands.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, monomeric enzyme that catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of cellular proteins. Product inhibition studies indicate that Nmt1p has an ordered Bi Bi reaction mechanism with myristoyl-CoA binding to the apo-enzyme to form a high affinity binary complex followed by binding of peptide with subsequent release of CoA and then the myristoylpeptide product. We have used isothermal titration calorimetry to quantify the effects of varying acyl chain length and removing the 3'-phosphate group of CoA on the energetics of interaction between Nmt1p and acyl-CoA ligands. Myristoyl-CoA binds to apo-Nmt1p with an affinity of 15 nM, corresponding to a binding free energy of -10.9 kcal/mol. This free energy is composed of a large favorable enthalpy of -24 kcal/mol and a large unfavorable entropic term. This large negative delta H degrees is consistent with a conformational change in the enzyme upon ligation, allowing synthesis of a functional peptide binding site. Binding of palmitoyl-CoA and lauroyl-CoA is driven by an exothermic enthalpy change which is much smaller than the corresponding parameter for myristoyl-CoA binding. The large differences in binding enthalpy and entropy (delta delta H degrees and T delta delta S degrees = 8-9 kcal/mol) demonstrate that the "off-length" acyl-CoAs bind to Nmt1p in a significantly different energetic fashion from myristoyl-CoA, even though the enzyme does not have a great deal of specificity among these ligands in terms of binding free energy (delta delta G degrees < or = 1 kcal/mol). The effect of removing the CoA 3'-phosphate group from myristoyl-CoA is similar to the effect of a two-carbon change in acyl chain length: i.e. an enthalpy dominated reduction in binding affinity. However, kinetic studies reveal that removing the 3'-phosphate from myristoyl-CoA has little effect on Nmt1p's catalytic efficiency, indicating that the 3'-phosphate group contributes binding free energy but little catalytic destabilization. The greater delta delta G degrees, with smaller delta delta H degrees and delta delta S degrees components, produced by removing the 3'-phosphate compared to increasing chain length suggests that it is not primarily the interactions of the 3'-phosphate which are disrupted when palmitoyl-CoA is substituted for myristoyl-CoA. No detectable interactions were noted between apo-Nmt1p and the substrate peptide, GAAPSKIV-NH2, providing additional support for the preferred ordered reaction mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)