4 research outputs found
Nuclear Localization Signals for Optimization of Genetically Encoded Tools in Neurons
Nuclear transport in neurons differs from that in non-neuronal cells. Here we developed a non-opsin optogenetic tool (OT) for the nuclear export of a protein of interest induced by near-infrared (NIR) light. In darkness, nuclear import reverses the OT action. We used this tool for comparative analysis of nuclear transport dynamics mediated by nuclear localization signals (NLSs) with different importin specificities. We found that widely used KPNA2-binding NLSs, such as Myc and SV40, are suboptimal in neurons. We identified uncommon NLSs mediating fast nuclear import and demonstrated that the performance of the OT for nuclear export can be adjusted by varying NLSs. Using these NLSs, we optimized the NIR OT for light-controlled gene expression for lower background and higher contrast in neurons. The selected NLSs binding importins abundant in neurons could improve performance of genetically encoded tools in these cells, including OTs and gene-editing tools.Peer reviewe
Optogenetic regulation of endogenous proteins
Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets. Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.Peer reviewe
Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change
pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin alpha (ProT alpha) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil-alpha-helix transition of ProTa and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid-liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe