Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs.

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.     

Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein.

The human and simian immunodeficiency virus envelope glycoproteins, which mediate virus-induced cell fusion, contain two putative amphipathic helical segments with large helical hydrophobic moments near their carboxyl-terminal ends. In an attempt to elucidate the biological role of these amphipathic helical segments, we have synthesized peptides corresponding to residues 768-788 and 826-854 of HIV-1/WMJ-22 gp160. Circular dichroism studies of the peptides showed that the alpha helicity of the peptides increased with the addition of dimyristoyl phosphatidylcholine (DMPC) indicating that the peptides form lipid-associating amphipathic helixes. The peptides solubilized turbid suspensions of DMPC vesicles, and electron microscopy of peptide-DMPC mixtures revealed the formation of discoidal complexes, suggesting that the peptides bind to and perturb lipid bilayers. The peptides were found to lyse lipid vesicles and caused carboxyfluorescein leakage from dye-entrapped egg phosphatidylcholine liposomes. The peptides also lysed human erythrocytes and were found to be toxic to cell cultures. At subtoxic concentrations, the peptides effectively inhibited the fusion of CD4+ cells infected with recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope proteins. Based on these results, and reported studies on the mutational analysis of HIV envelope proteins, we suggest that the amphipathic helical segments near the carboxyl terminus of HIV envelope proteins may play a role in lysis of HIV-infected cells and also may modulate the extent of cell fusion observed during HIV infection of CD4+ cells.     

H-Type Bovine Spongiform Encephalopathy: Complex Molecular Features and Similarities with Human Prion Diseases

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrP^res) in Western blot, with a 1–2 kDa higher apparent molecular mass of the unglycosylated PrP^res associated with labelling by antibodies against the 86–107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrP^res, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrP^res (PrP^res #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrP^res fragment of ≈7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrP^res #2 and ≈7 kDa PrP^res were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.Key Words: prion, BSE, Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker, Western blot, amyloid     

<Emphasis Type="Italic">M. tuberculosis</Emphasis> genotypic diversity and drug susceptibility pattern in HIV- infected and non-HIV-infected patients in northern Tanzania

Background  

Tuberculosis (TB) is a major health problem and HIV is the major cause of the increase in TB. Sub-Saharan Africa is endemic for both TB and HIV infection. Determination of the prevalence of M. tuberculosis strains and their drug susceptibility is important for TB control.     

Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.     

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.     

Anti-inflammatory and cholesterol-reducing properties of apolipoprotein mimetics: a review

Reduced levels of HDL cholesterol (HDL-C) are a strong independent predictor of coronary artery disease (CAD) risk. The major anti-atherogenic function of HDL is to mediate reverse cholesterol transport. This response is highly dependent on apoA-I and apoE, protein components of HDL. Randomized clinical trials have assessed effects of several classes of drugs on plasma cholesterol levels in CAD patients. Agents including cholestyramine, fibrates, niacin, and statins significantly lower LDL cholesterol (LDL-C) and induce modest increases in HDL-C, but tolerance issues and undesirable side effects are common. Additionally, residual risk may be present in patients with persistently low HDL-C and other complications despite a reduction in LDL-C. These observations have fueled interest in the development of new pharmacotherapies that positively impact circulating lipoproteins. The goal of this review is to discuss the therapeutic potential of synthetic apolipoprotein mimetic peptides. These include apoA-I mimetic peptides that have undergone initial clinical assessment. We also discuss newer apoE mimetics that mediate the clearance of atherogenic lipids from the circulation and possess anti-inflammatory properties. One of these (AEM-28) has recently been given orphan drug status and is undergoing clinical trials.     

High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction  

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.