scholarly journals All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

2021 ◽  
Vol 118 (36) ◽  
pp. e2105548118
Author(s):  
Aitor Franco ◽  
Pablo Gracia ◽  
Adai Colom ◽  
José D. Camino ◽  
José Ángel Fernández-Higuero ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
High Speed ◽  
Amyloid Formation ◽  
Kinetic Characterization ◽  
Force Microscopy ◽  
Toxic Species ◽  
Disassembly Process ◽  
Atomic Force ◽  
Selective Processing

α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

DESIGN AND CHARACTERIZATION OF A NOVEL SCANNER FOR HIGH-SPEED ATOMIC FORCE MICROSCOPY

2006 ◽  
Vol 39 (16) ◽  
pp. 819-824 ◽  
Author(s):  
Georg Schitter ◽  
Georg E. Fantner ◽  
Philipp J. Thurner ◽  
Jonathan Adams ◽  
Paul K. Hansma
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Force Microscopy ◽  
Atomic Force

scholarly journals Characterization of the motion of membrane proteins using high-speed atomic force microscopy

2012 ◽  
Vol 7 (8) ◽  
pp. 525-529 ◽  
Author(s):  
Ignacio Casuso ◽  
Jonathan Khao ◽  
Mohamed Chami ◽  
Perrine Paul-Gilloteaux ◽  
Mohamed Husain ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Membrane Proteins ◽  
High Speed ◽  
Force Microscopy ◽  
Atomic Force

scholarly journals On the Determination of Mechanical Properties of Aqueous Microgels—Towards High-Throughput Characterization

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 64
Author(s):  
Ingrid Haga Oevreeide ◽  
Renata Szydlak ◽  
Marcin Luty ◽  
Husnain Ahmed ◽  
Victorien Prot ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
High Throughput ◽  
High Speed ◽  
Soft Matter ◽  
Force Microscopy ◽  
Fluid Field ◽  
Atomic Force ◽  
Fundamental Point

Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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