Dataset Information

Key Residues for the Formation of A?42 Amyloid Fibrils.

ABSTRACT: Formation of amyloid fibrils by A?42 protein is a pathological hallmark of Alzheimer's disease. A?42 fibrillization is a nucleation-dependent polymerization process, in which nucleation is the rate-limiting step. Structural knowledge of the fibril nucleus is important to understand the molecular mechanism of A? aggregation and is also critical for successful modulation of the fibrillization process. Here, we used a scanning mutagenesis approach to study the role of each residue position in A?42 fibrillization kinetics. The side chain we used to replace the native residue is a nitroxide spin label called R1, which was introduced using site-directed spin labeling. In this systematic study, all residue positions of A?42 sequence were studied, and we identified six key residues for the A?42 fibril formation: H14, E22, D23, G33, G37, and G38. Our results suggest that charges at positions 22 and 23 and backbone flexibilities at positions 33, 37, and 38 play key roles in A?42 fibrillization kinetics. Our results also suggest that the formation of a ?-strand at residues 15-21 is an important feature in A?42 fibril nucleus. In overall evaluation of all of the mutational effects on fibrillization kinetics, we found that the thioflavin T fluorescence at the aggregation plateau is a poor indicator of aggregation rates.

SUBMITTER: Hsu F

PROVIDER: S-EPMC6068601 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Key Residues for the Formation of Aβ42 Amyloid Fibrils.

Hsu Frederick F Park Giovanna G Guo Zhefeng Z

ACS omega 20180701 7

Formation of amyloid fibrils by Aβ42 protein is a pathological hallmark of Alzheimer's disease. Aβ42 fibrillization is a nucleation-dependent polymerization process, in which nucleation is the rate-limiting step. Structural knowledge of the fibril nucleus is important to understand the molecular mechanism of Aβ aggregation and is also critical for successful modulation of the fibrillization process. Here, we used a scanning mutagenesis approach to study the role of each residue position in Aβ42 ...[more]

PMID: 30087945

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Project description:Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Dataset Information

Key Residues for the Formation of A?42 Amyloid Fibrils.

Publications

Key Residues for the Formation of Aβ42 Amyloid Fibrils.

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