Enhanced sensitivity of mechano-sensors and their coupling to the signaling

Enhancement of cell sensitivity to mechnostimuli by the ectopic expression of mechanosensing ion channels and gas vesicles.

Gas vesicles sensitize cells to respond to ultrasound which triggers opening of mechanosensitive calcium channels. Influx of free calcium ions activates the calcium-responsive reporters and reconstitutes a mediator to activate the downstream signaling pathway.

The challenge of increasing the sensitivity of cells to ultrasound or other mechanic stimuli could be solved by overexpression of ultrasound responsive ion channels. Mechanosensitive promoters have been investigated by the MIT 2010 team, however we were interested in mechanosensitive receptors. There are reports in the literature that some TRP family mammalian channels and bacterial channels (MscS and MscL) are responsive to osmotic shock, which led us to hypothesize that they should also be able to respond to ultrasound. An additional idea for the enhancement of the mechanosensors occurred to us through browsing through some previous iGEM projects. Protein gas vesicles have been used in bacteria by several iGEM teams (Melbourne 2007, Groningen 2009 , UCL2012, Glasgow2014) and OUC-China team reported in 2012 that they could reconstitute gas vesicles in E. coli from only two components. Gas vesicles, in contrast to cytosol, are compressible, so application of pressure waves to the gas vesicles should strongly affect the cellular cytoskeleton and the membrane of cells containing such gas vesicles. If we could successfully reconstitute gas vesicles in mammalian cells this could strongly increase the response of cells to mechanical stimuli and ultrasound.

Activation of mechanosensing receptors usually leads to an influx of calcium ions into cells, which might thereby be coupled to reporters or selected signaling pathways via formation of a calcium dependent complex. Several calcium responsive protein probes have been reported in the past, such as those based on calmodulin, and we set out to identify the design that would efficiently respond within the range of calcium ion concentrations triggered by mechanosensor stimulation (1).

 Proteolysis based signaling pathway and logic function processing

Our idea was to use highly specific proteolysis to mediate protein signaling. In comparison to phosphorylation, proteolysis is irreversible, but could nevertheless lead to a response as fast as the one based on phosphorylation, regardless of the longer time to replenish the degraded proteins once the signal is no longer present. This is in fact sufficient for most envisioned therapeutic applications. Cleavage of a protein can either lead to activation or inactivation of the selected protein and could be engineered by introduction of a target cleavage site into the protein of interest. Some proteases have highly specific recognition sites, such as for example thrombin or the Tobacco etch virus protease (TEVp). This high specificity prevents toxicity of activated proteases to cells due to degradation of essential proteins and activation of cell death. To use proteases as signal mediators, their activation would have to be triggered by a signal. We reasoned this could be achieved by reconstitution of split proteases, either by an external signal or by cleavage of peptides preventing reconstitution. The latter would also allow the engineering of a cascade of proteolytic activity with each enzyme activating a downstream protein with an introduced specific target site, functioning as a linear signaling pathway.

Site specific orthogonal split proteases can be used to design a protease-based signaling pathway or a complex logic function processing cellular circuit.

Proteases can also trigger the controlled rapid release of protein from endoplasmic reticulum.

Split proteases could also be used to combine several input signals. To test this hypothesis we would need to expand the toolbox of highly specific proteases (similar to TEVp) that should be highly orthogonal and harmless to cells, design appropriate split variants of these proteases and design a system where proteolytic cleavage results in activation. Such a system was previously designed based on competition between linked coiled-coil domains, however it has only been reported in vitro Shekhawat2009. Furthermore, the reported system was not based on split protease reconstitution and was therefore not responsive to external inputs.

Finally, we realized that site specific proteolysis could also be used to solve the challenge of the release of molecules from cells. Secretion of proteins from cells occurs via the ER and the secretory pathway, while the retention of proteins in the ER occurs via the ER retrieval signals. Proteolytic cleavage of the retrieval peptide could release the selected cargo proteins or peptides into the secretory pathway, and result in protein secretion from the pool of already synthesized proteins (2).