Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.
The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA–protein conjugation still limit true emulation of natural host–guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA–protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host–guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.
Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance-dependent energy transfer between two preloaded fluorophores. Interestingly, single-molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine-tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.
Die Methoden der DNA-Nanotechnologie gehçren heutzutage zu den am weitesten fortgeschrittenen Verfahren zur räumlich und zeitlich kontrollierten Manipulation chemischer Systeme im Nanometerbereich. In der vorliegenden Arbeit konnten wir durch eine Kombination von DNA-Origami-Strukturen, welche räumlich definiert adressiert werden kçnnen, mit schaltbaren und dabei mechanische Bewegung auslçsenden DNA-Motiven neuartige dynamisch rekonfigurierbare DNA-Nanokapselsysteme erzeugen. Die Dynamik des Systems wurde dabei durch eine reversible Streckung bzw. Kontraktion der zentralen Kavität in der DNA-Nanostruktur erzeugt und konnte mittels eines distanzabhängigen Energietransfers zwischen zwei in der Struktur integrierten Fluorophoren über FRET-Einzelmolekülanalysen gemessen werden. Interessanterweise zeigten diese Messungen, dass die Kinetik der einzelnen Umlagerungsprozesse stark von den verwendeten Strukturmotiven und/oder Aktuatorsequenzen abhängig war, wodurch eine besondere Eignung von FRET-Einzelmolekülanalysen zur Optimierung der Dynamikeigenschaften großer DNA-Nanostrukturen unterstrichen wird. Dabei glauben wir, dass die hier gezeigten DNA-Nanosysteme in der Zukunft als Modellstrukturen für artifizielle, biomimetische Kompartimente oder Transportsysteme dienen kçnnten.
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