Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH.

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson's disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Structure of the AcrAB-TolC multidrug efflux pump.

The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.

Maturation-dependent changes in the size, structure and seeding capacity of Aß42 amyloid fibrils.

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-ß network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-ß sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species.

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

Correction to 'Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species'.

[This corrects the article DOI: 10.1098/rsos.171399.].