ABSTRACT
Neurotoxic Aß42 oligomers are believed to be the main cause of Alzheimer's disease. Previously, we found that the C-terminal fragments (CTFs), Aß(30-42) and Aß(31-42) were the most potent inhibitors of Aß42 oligomerization and toxicity in a series of Aß(x-42) peptides (x=28-39). Therefore, we chose these peptides as leads for further development. These CTFs are short (12-13 amino acids) hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the Nâ terminus resulted in toxic peptides. Therefore, we next incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting the ß-sheet formation. Focusing on Aß(31-42), we used a two-step N-methyl amino acid substitution strategy to study the structural factors controlling inhibition of Aß42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side chain. This strategy allowed exploring the role of both side chain structure and N-Me substitution in inhibitory activity. We found that the introduction of an N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aß(31-42). In particular, N-Me amino acid substitution at position 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aß42 toxicity by short peptides is highly structure-specific, providing a basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Alzheimer Disease/drug therapy , Amino Acid Sequence , Amyloid beta-Peptides/metabolism , Animals , Cell Survival/drug effects , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , PC12 Cells , Peptide Fragments/pharmacology , Peptide Fragments/toxicity , Protein Engineering , Protein Structure, Secondary , Rats , Solubility , Structure-Activity RelationshipABSTRACT
BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses--with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses. STUDY DESIGN AND METHODS: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization. RESULTS: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed. CONCLUSION: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism.
Subject(s)
Blood Donors , High-Throughput Screening Assays/methods , Leukemia Virus, Murine/isolation & purification , Neutralization Tests/methods , Xenotropic murine leukemia virus-related virus/isolation & purification , Animals , Antibodies, Neutralizing , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Blood Donors/statistics & numerical data , Cell Line, Tumor , Female , HEK293 Cells , Humans , Leukemia Virus, Murine/immunology , Macaca mulatta , Male , Mice , Microchemistry/methods , NIH 3T3 Cells , Retroviridae Infections/blood , Retroviridae Infections/diagnosis , Retroviridae Infections/epidemiology , Retroviridae Infections/virology , Xenotropic murine leukemia virus-related virus/immunologyABSTRACT
Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.
