The mechanism of prion protein (PrP) conversion, the key event in prion diseases, is still not understood. We investigated how perturbations of interactions between the subdomains β1-α1-β2 and α2-α3 affect PrP conversion. In vitro fibrillization and biophysical methods were used to relate mouse PrP conversion kinetics to thermodynamic stability. We show that pathologic mutations H187R and E196K destabilize PrP and accelerate fibrillization. At acidic pH, the major contribution to the destabilization of PrP comes from the protonation of histidine 187 because its replacement by tyrosine led to more stable protein with slower fibrillization. Furthermore, we show that the introduction of a novel histidine residue into the subdomain interface (F198H) acts as a pH-inducible switch that promotes conversion upon histidine protonation, whereas this effect is not observed when His residue is introduced at the protein surface (Y155H). We observed a strong correlation between the stability of native structure and kinetics of fibrillization of PrP variants. Our results show that pathologic mutations promote subdomain separation and suggest that stabilization of the native structure might be a viable strategy for the development of novel therapeutics for prion diseases.
COBISS.SI-ID: 5610266
Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA binding domains. Here, we employed the transcription activator–like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information processing devices.
COBISS.SI-ID: 5408026
Electroporation is a frequently used method for increasing permeability of the cell membrane, but can also cause damage to the cell: cytosol leakage, ROS formation, osmotic swelling, necrosis and induction of apoptosis. In this study we analysed weather electroporation of macrophages in vitro can trigger NLRP3 inflammasome activation and subsequent secretion of IL-1β. Cells were exposed to electric pulses and quantity of secreted IL-1β was determined. Our results show that the observed IL-1β secretion was not NLRP3 inflammasome dependent but indicate that nevertheless, electroporation triggers a proinflammatory immune response through IL-1β secretion.
COBISS.SI-ID: 10780500