Biologically active analogs of thymopentin with enhanced enzymatic stability

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32–36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro²-TP5-NH₂ and Aib²-TP5-NH₂ retained activity and were shown to exhibit a high degree of stability when incubated in human serum.     

Biologically active loop-shaped analogs of bradykinin and polisteskinin

The classical methods of peptide chemistry have been employed to synthesize loop-shaped derivatives of bradykinin and polisteskinin, Lys-Lys-Lys-[cyclo (9----1 epsilon), Lys1, Gly6]bradykinin and Lys-Lys-Lys-Leu-Arg-Gly[cyclo (9----1 epsilon)Lys1, Gly6] bradykinin. In the course of synthesis, the linear "tail" fragments were attached to partially deblocked cyclopeptide. Protective groups were removed by treating with hydrogen fluoride, the end products were purified using reversed-phase and ion exchange chromatography. Biological experiments in vivo have revealed that the two compounds elicit a prolonged hypotensive effect in rats which is characteristic of cyclic bradykinin analogues. With the latter compound, a decrease in arterial pressure is preceded by a brief hypertensive action. The loop-shaped analogues are slightly myotropic when applied to rat uterus preparations in vitro.     

Biologically active conformations of thymopentin. Studies with conformationally restricted analogs

Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.     

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.     

The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids

The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.     

Study of a series of analogs of salmon calcitonin in which alanine replaces leucine

Leucine residues at positions 12, 16 and 19 of salmon calcitonin were systematically replaced by alanine either one, two or three at a time. Substitution of Ala at positions 12 or 16 had the greatest effect on the structure of the peptide and on the ability of the peptide to attain structures of higher helical content in the presence of lipid. Despite the similar effects on the conformational properties, the effects on activity are markedly different for analogs with Ala substitutions at positions 12 and 16. The Ala12 derivatives are much less active while the Ala16 derivatives are slightly more active than the parent hormone. In contrast to the effects of substitutions at these positions, substitution of Ala at position 19 has relatively little effect on activity or on conformation. These results demonstrate that different regions of the calcitonin molecule have different conformational requirements for maximal activity.     

Angiotensin and bradykinin interactions with phospholipids

Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp¹)-angiotensins. The apparent dissociation constant of this complex in chloroform was 10^?5 M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behavior of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp¹)-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo.     

Cholesterol interactions with phospholipids in membranes.

Mammalian cell membranes are composed of a complex array of glycerophospholipids and sphingolipids that vary in head-group and acyl-chain composition. In a given cell type, membrane phospholipids may amount to more than a thousand molecular species. The complexity of phospholipid and sphingolipid structures is most likely a consequence of their diverse roles in membrane dynamics, protein regulation, signal transduction and secretion. This review is mainly focused on two of the major classes of membrane phospholipids in eukaryotic organisms, sphingomyelins and phosphatidylcholines. These phospholipid classes constitute more than 50% of membrane phospholipids. Cholesterol is most likely to associate with these lipids in the membranes of the cells. We discuss the synthesis and distribution in the cell of these lipids, how they are believed to interact with each other, and what cellular consequences such interactions may have. We also include a discussion about findings in the recent literature regarding cholesterol/phospholipid interactions in model membrane systems. Finally, we look at the recent trends in computer and molecular dynamics simulations regarding phospholipid and cholesterol/phospholipid behavior in bilayer membranes.     

Angiotensin and bradykinin interactions with phospholipids.

Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp1)-angiotensins. The apparent dissociation constant of this complex in chloroform was 10(-5) M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behavior of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp1)-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo.     

Calcium- and calmodulin-sensitive interactions of calcineurin with phospholipids

Physical association of calcineurin with phosphatidylserine (PS) or phosphatidylglycerol (PG) was observed by molecular exclusion chromatography; the enzyme did not associate with phosphatidylethanolamine or phosphatidylcholine. The interactions with PS and PG were enhanced by Ca2+ which implicates a regulatory role for the Ca2+-binding subunit in this process. Addition of PG or PS to standard calcineurin assays elicited profound changes in enzymatic activity; phosphatidylcholine and phosphatidylethanolamine were without effect. Up to 23-fold stimulation of the calmodulin-independent activity was observed with phosphorylated histone H1 or synapsin I as the substrates. In contrast, the activity toward p-nitrophenyl phosphate and tyrosine phosphate was found to be inhibited. A characterization and comparison of the two opposite responses showed that: the phospholipids had insignificant effects on the Km for substrates, the phospholipid specificity for activation and inhibition was nearly indistinguishable, half-maximal activation and inhibition were obtained at similar concentrations of PG (K0.5 = 0.21 and 0.14 mg/ml, respectively), and calmodulin enhanced the responses to PG (K0.5 = 0.064 and 0.033 mg/ml for activation and inhibition, respectively) to similar extents. Together, these observations demonstrate that the two substrate-dependent responses of calcineurin are due to the association of the phosphatase with phospholipids and not a result of substrate-phospholipid interactions. This suggests that Ca2+- and calmodulin-stimulated interactions of calcineurin with acidic phospholipids may play a role in regulating the substrate specificity of this multifunctional phosphatase.     

Biologically active peptides interacting with the G protein-coupled formylpeptide receptor

Leucocytes accumulate at sites of inflammation and microbial infection in response to locally produced chemotactic factors. N-formylpeptides produced by Gram negative bacteria were among the first chemotactic factors structurally defined which signal through G protein-coupled formylpeptide receptor (FPR) and FPR-like 1 (FPRL1) expressed by phagocytic leukocytes in human and in mouse homogogues mFPR and mFPR2. During the past few years, a number of pathogen- and host-derived agonists/antagonists for FPR, FPRL1 and another FPR variant FPR-like 2 (FPRL2) have been identified. Activation of formylpeptide receptors (FPRs) in phagocytic leukocytes by agonists results in increased cell chemotaxis, phagocytosis, and release of pro-inflammatory mediators. Peptide agonists for FPRs have also been shown to possess immune adjuvant activity when injected in mice. In addition, FPR aberrantly expressed on highly malignant human glioblastoma cells promotes tumor cell migration, proliferation and production of vascular endothelial growth factor in response to agonists released by necrotic tumor cells. Therefore, formylpeptide receptor ligands, by interacting with FPRs, play important roles in host defense and in the rapid progression of human glioblastoma.     

Fluorimetric studies of protein kinase C interactions with phospholipids

Dansyl-phosphatidylserine (D-PS) was used as a fluorescence probe to study interactions between protein kinase C (PKC) with phospholipid vesicles. It was found that D-PS fluorescence (520 nm) was enhanced by PKC (excited at 285 nm). The fluorescence energy transfer, indicative of a close association of PKC with D-PS vesicles, was differentially modulated by various phospholipids, depending upon their effects on PKC activation state and the manners in which they were present. PKC inhibitors (e.g. polymyxin B and ether lipids) potently inhibited the PKC-enhanced D-PS fluorescence. It is suggested that certain spatial arrangements between PKC and its phospholipid cofactor are essential for the enzyme activation and that D-PS would be a useful probe to study fluorimetrically such interactions.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Ras GTPase-activating protein physically associates with mitogenically active phospholipids.

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.     

Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors.

We have constructed retroviral expression vectors by manipulation of the Moloney murine leukemia virus genome such that an exogenous DNA sequence may be inserted and subsequently expressed when introduced into mammalian cells. A series of N-terminal deletions of the v-mos oncogene was constructed and assayed for biological activity with these retroviral expression vectors. The results of the deletion analysis demonstrate that the region of p37mos coding region upstream of the third methionine codon is dispensable with respect to transformation. However, deletion mutants of v-mos which allow initiation of translation at the fourth methionine codon have lost the biological activity of the parental v-mos gene. Furthermore, experiments were also carried out to define the C-terminal limit of the active region of p37mos by the construction of premature termination mutants by the insertion of a termination oligonucleotide. Insertion of the oligonucleotide just 69 base pairs upstream from the wild-type termination site abolished the focus-forming ability of v-mos. Thus, we have shown the N-terminal limit of the active region of p37mos to be between the third and fourth methionines, while the C-terminal limit is within the last 23 amino acids of the protein.     

Are interactions with phospholipids responsible for pharmacological activities of cardiotoxins?

Cardiotoxins are small basic proteins (7 000 daltons) that are found in the venoms of Elapidae snakes. Although they are structurally close to alpha-neurotoxins present in the same secretions, their activity is related to their ability to interact with every cell membrane inducing, near micromolar concentration, the modification of its biological properties and/or physical structure. The mode of action of cardiotoxins, on a molecular level, is still under investigation. However, lipid-protein interactions are more and more involved in their binding to membrane and in their activities. Using new experimental data a better definition of phospholipid-cardiotoxin interaction is arrived at and a tentative molecular explanation of the pharmacological activities of these proteins is presented and discussed.     

Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).