Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly 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 non-toxic 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 non-toxic 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.
We report the development of a high-level bacterial expression system for the Alzheimer’s disease-associated amyloid β-peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Aβ(1–40) and Met-Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.
A number of phosphatidylcholine (PC) cations spanning a mass range of 400 to 1000 Da are investigated using electrospray ionization mass spectrometry coupled with traveling wave ion mobility spectrometry (TWIMS). A high correlation between mass and mobility is demonstrated with saturated phosphatidylcholine cations in N2. A significant deviation from this mass-mobility correlation line is observed for the unsaturated PC cation. We found that the double bond in the acyl chain causes a 5% reduction in drift time. The drift time is reduced at a rate of ~1% for each additional double bond. Theoretical collision cross sections of PC cations exhibit good agreement with experimentally evaluated values. Collision cross sections are determined using the recently derived relationship between mobility and drift time in TWIMS stacked ring ion guide (SRIG) and compared to estimate collision cross-sections using empiric calibration method. Computational analysis was performed using the modified trajectory (TJ) method with nonspherical N2 molecules as the drift gas. The difference between estimated collision cross-sections and theoretical collision cross-sections of PC cations is related to the sensitivity of the PC cation collision cross-sections to the details of the ion-neutral interactions. The origin of the observed correlation and deviation between mass and mobility of PC cations is discussed in terms of the structural rigidity of these molecules using molecular dynamic simulations.
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