Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo

Amyloid β-protein (Aβ) self-assembly into toxic oligomers and fibrillar polymers is believed to cause Alzheimer’s disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met35 to sulfoxide has been reported to decrease Aβ assembly and neurotoxicity, whereas surprisingly, Met35 oxidation to sulfone yields similar toxicity to unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region, but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA−/− mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to non-transgenic mice. Immunization with a novel methionine sulfoxide-rich antigen for six months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD

A Key Role for Lysine Residues in Amyloid β-Protein Folding, Assembly, and Toxicity

A combination of hydrophobic and electrostatic interactions is important in initiating the aberrant self-assembly process that leads to formation of toxic oligomers and aggregates by multiple disease-related proteins, including amyloid β-protein (Aβ), whose self-assembly is believed to initiate brain pathogenesis in Alzheimer’s disease. Lys residues play key roles in this process and participate in both types of interaction. They also are the target of our recently reported molecular tweezer inhibitors. To obtain further insight into the role of the two Lys residues in Aβ assembly and toxicity, here we substituted each by Ala in both Aβ40 and Aβ42 and studied the impact of the substitution on Aβ oligomerization, aggregation, and toxicity. Our data show that each substitution has a major impact on Aβ assembly and toxicity, with significant differences depending on peptide length (40 versus 42 amino acids) and the position of the substitution. In particular, Lys16→Ala substitution dramatically reduces Aβ toxicity. The data support the use of compounds targeting Lys residues specifically as inhibitors of Aβ toxicity and suggest that exploring the role of Lys residues in other disease-related amyloidogenic proteins may help understanding the mechanisms of aggregation and toxicity of these proteins

C-Terminal Tetrapeptides Inhibit Aβ42-Induced Neurotoxicity Primarily through Specific Interaction at the N-Terminus of Aβ42

Inhibition of amyloid β-protein (Aβ)-induced toxicity is a promising therapeutic strategy for Alzheimer’s disease (AD). Previously, we reported that the C-terminal tetrapeptide Aβ(39–42) is a potent inhibitor of neurotoxicity caused by Aβ42, the form of Aβ most closely associated with AD. Here, initial structure–activity relationship studies identified key structural requirements, including chirality, side-chain structure, and a free N-terminus, which control Aβ(39–42) inhibitory activity. To elucidate the binding site­(s) of Aβ(39–42) on Aβ42, we used intrinsic tyrosine (Y) fluorescence and solution-state NMR. The data suggest that Aβ(39–42) binds at several sites, of which the predominant one is located in the N-terminus of Aβ42, in agreement with recent modeling predictions. Thus, despite the small size of Aβ(39–42) and the hydrophobic, aliphatic nature of all four side-chains, the interaction of Aβ(39–42) with Aβ42 is controlled by specific intermolecular contacts requiring a combination of hydrophobic and electrostatic interactions and a particular stereochemistry

Tranilast Binds to Aβ Monomers and Promotes Aβ Fibrillation

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer’s disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ–tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients

Molecular Tweezers Inhibit Islet Amyloid Polypeptide Assembly and Toxicity by a New Mechanism

In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet β-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)­peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe <i>in vivo</i>, supporting its development toward disease-modifying therapy for T2D

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins

Author Manuscript 2012 October 26.Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein selfassembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β- protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.University of California, Los Angeles. (Jim Easton Consortium for Alzheimer’s Drug Discovery and Biomarker Development)American Health Assistance Foundation (Grant A2008-350)National Institutes of Health (U.S.) (National Institute on Aging Grant AG027818