In publication Fink Fink et al. 2019 Nature Chemical Biology we published results describing the use of orthogonal coiled coil forming peptides for fast signalization. Even though we do not yet fully understand the inner workings of cells, we are able to modify cells to allow them to respond to specific extracellular signals, which is a crucial feature for the use of cells in therapeutic applications. The implementation of novel control systems inside cells is an important goal of synthetic biology. With orthogonal coiled coil peptides linked to functional elements the directed and carefully regulated cellular activity was achieved. The faster response, within minutes instead of hours, was achieved by a careful design of interactions between proteins that can be cleaved into smaller fragments that led them to assemble into new complexes as a response to input signals. This discovery could represent a valuable tool for the development of future medical and biotechnological applications.
COBISS.SI-ID: 39867909
TTo construct protein/polypeptide structures, that may represent carriers of drugs, we used modular strategy that exploites coiled-coil (CC) dimers, orthogonal peptide pairs forming coiled coils, as building modules. The 3D structure is defined by long range long-range interactions between pairs of modules arranged in a polypeptide chain in a defined order. In this study we present a de novo design of second-generation CC protein 3D structures, based on a toolbox of supercharged CC dimer building modules and on a computational design platform, which enabled the construction of protein structures that folded efficiently in vivo. Second-generation designs were also produced that self-assembled without the need for refolding in bacteria, in mammalian cells, and in mice without causing inflammation or other adverse pathological effects, thus opening a path toward new applications of designed protein cages.
COBISS.SI-ID: 6266906
Natural activators of receptor TLR9 are bacterial and viral DNA. For the design of adjuvants for vaccines and in immunotherapy, it is important to identify an optimal nucleotide sequence of synthetic agonists that enable activation of target human cells. Analysis of required oligonucleotide motives of TLR9 agonists showed that minimal required motifs activating mouse TLR9 differ from that required for activation of human TLR9. We showed that only oligonucleotides with two spatially positioned CpG motives could activate the human antigen presenting cells like plasmacytoid dendritic cells and B-cells. Moreover, we showed that these stringent requirements for the synthetic agonists are consequences of the three amino acid residues within human TLR9. New discoveries are important for the human vaccine development using TLR9 agonists as adjuvants (also for immunotherapy). The vaccines are tested on mice during the development stages; therefore it is critical to consider the identified differences in minimal sequence requirements for activation of mouse and human TLR9. Taken together, the discoveries are important for understanding the molecular mechanism of TLR9 activation and for the design of new agonists as potential therapeutic drugs.
COBISS.SI-ID: 39287301
Flagellin, the main protein component of bacterial flagella, is an agonist of membrane TLR5 receptor and cytosolic NLRC4 receptor. Flagellin is the only protein agonist of TLR receptors and is being already tested as an adjuvant for modern component vaccines. In the described study, we identified segments of the flagellin of Salmonella and Serratia, which are important for the stabilization of the TLR5 : receptor active form. The findings were confirmed by the chimeric flagellins that combine the segments of the Helicobacter pylori and Salmonella typhimurium flagellin. We confirmed that the D1 domain of flagellin is important for binding to the receptor and the D0 domain, in particular the C-terminal part, is essential for the activation of TLR5. Membrane receptor TLR5 is crucial for identifying bacterial infections and activation of innate immunity. Activation of the receptor is also important for the activation of the acquired immunity. The discovery is important for the human vaccine development using TLR5 agonists as adjuvants (also for immunotherapy).
COBISS.SI-ID: 6207514