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The level of calmodulin increases in cells expressing HIV-1 envelope glycoprotein. Although a calmodulin increase is bound to alter many cellular metabolic and signaling pathways, the benefits to the virus of these alterations must be indirect. However, the possibility exists that increased cellular calmodulin benefits the virus by directly associating with nonenvelope viral proteins. We have, therefore, investigated whether calmodulin can interact with HIV structural proteins Gag, p17, and p24. Calmodulin binds Gag and p17 but not p24 in (125)I-labeled calmodulin overlays of SDS-polyacrylamide gels. Removal of calcium by addition of EGTA eliminates this binding. A computer algorithm for predicting helical regions that should bind calmodulin predicts that there are two calmodulin-binding regions near the N terminus of p17. Intrinsic tryptophan fluorimetry shows that two peptides, each of which includes one of the predicted regions, bind calmodulin: p17(11-25) binds calmodulin with a 2-to-1 stoichiometry and dissociation constant of approximately 10(-9) M(2), and p17(31-46) also binds calmodulin with a dissociation constant of about 10(-9) M. These binding sites are nearly contiguous, forming an extended calmodulin-binding domain p17(11-46). In H-9 cells, Gag and calmodulin colocalize within the resolution of confocal light microscopy.

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Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.

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We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.

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Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

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Trypanosoma brucei brucei is non-infectious to man due to the sensitivity of these parasites to the lytic activity of normal human serum. Apolipoproteins (apo) have been purified, under non-denaturing conditions, from the subclass of human high-density lipoprotein (HDL), termed trypanosome lytic factor (TLF), which is responsible for the cytotoxicity of human serum to T. b. brucei. The TLF apolipoproteins were purified by anion exchange chromatography in the presence of the nonionic detergent octylglucoside and a reconstitution method was developed which allowed the role of the individual apolipoproteins and different lipids to be assessed. The results suggest that the TLF lipids do not have a direct role in lysis but are necessary for the correct assembly of the lytic HDL particle. Apo A-I, apo L-III and apo L-I contribute to lysis in reconstituted particles but individually they are not cytotoxic. Apo A-II was not required in the reconstituted TLF particle for trypanosome lysis. Formation of a lytic HDL particle required apo L-III suggesting its potential role as a toxin. Thermal inactivation of TLF activity correlated with the amount of denatured apo L-I, indicating that apo L-I was involved in lysis of T. b. brucei by native TLF.

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The appearance of anionic lipids on the extracellular surface of cells is required for the formation of the procoagulant complex that leads to the activation of prothrombin. Procoagulant activity would be expected to be inhibited by substances that stabilize the membrane structure and hence inhibit the transbilayer diffusion of phosphatidylserine from the cytoplasmic to the extracellular surface of the plasma membrane. The generation of procoagulant activity in human erythrocytes by A23187 and Ca2+ is inhibited by apolipoprotein A-I, its amphipathic peptide analogues, and high-density lipoprotein (HDL). These agents do not inhibit the Ca2+ loading of erythrocytes by A23187, nor do they inhibit the activation of prothrombin once the cells have been incubated at 37 degrees C with A23187 and Ca2+. Transbilayer diffusion of fluorescently labeled phosphatidylserine is inhibited by apolipoprotein A-I. These findings indicate that class A amphipathic helixes as well as lipoprotein particles and liposomes inhibit the transbilayer diffusion of phospholipids and procoagulant activity. This activity may contribute to the protective role of HDL against arteriosclerosis and thrombosis.

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The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF-mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.

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Apolipoprotein (class A) amphipathic helixes are postulated to act as detergents by virtue of their cross-section being wedge-shaped. Using computer analysis of naturally occurring class A and lytic (class L) amphipathic helixes, we designed two archetypical model peptides. Analogs of these two peptides, incorporating substitutions or modifications of interfacial or basic residues, had the following effects. Class A peptides stabilized bilayer structure, reduced leakage from large unilamellar vesicles and erythrocytes, and inhibited lysis induced by class L peptides. Class L peptides destabilized bilayer structure in model membranes and increased binding of class A peptides to erythrocytes. The ability of class L analogs to lyse membranes and induce inverted lipid phases was reduced by either decreasing the bulk of an interfacial residue, increasing the angle subtended by the polar face, or increasing the bulk of the basic residues. The ability of the class A analog to stabilize bilayer structure and inhibit erythrocyte lysis by class L peptides was enhanced by methylating the Lys residues. These results can be explained by a model that we term the reciprocal wedge hypothesis. By analogy to the reciprocal effects of phospholipid shapes on membrane structure, we propose that the wedge shape of class A helixes stabilizes membrane bilayers, whereas the inverted wedge shape of class L helixes destabilizes membrane bilayers, and, thus, one class will neutralize the effect of the other class on membranes.

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In a recent classification of biologically active amphipathic alpha-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G., Anantharamaiah, G.M. Proteins 8:103-117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the alpha-amino terminus and an amide at the alpha-carboxyl terminus, to obtain Ac-18A-NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the alpha-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC-water interface is increased only by about 3% (from -8.0 kcal/mol for 18A to -8.3 kcal/mol for Ac-18A-NH2). The Ac-18A-NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH2 more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles.

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Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.

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Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO2 in the 'dark' reactions of photosynthesis. The importance of these findings is discussed.