standard Borrowing Signaling Pathways from Bacteria for Use in Eukaryotes

Using genetic technologies for constructing signaling pathways in vivo opens the door to sophisticated manipulations of the host organism and Hansen et al (2014) in a recent issue of PNAS describe their successful transplantation of three two-component signaling pathways from E. coli into mammalian cells.

A generic 2-component system with a membrane sensor histidine kinase (HK).

A generic 2-component system with a membrane sensor histidine kinase (HK).

Their results beg the question as to whether these systems could be used in transgenic insects.

Already available to insect biologists are simple one-component transcription systems such as Gal4 /UAS, Q/QUAS and Tet-on/Tet-off. You express the transcription factor, for example Gal4, and it turns on a target transgene under the regulatory control of a promoter with the appropriate transcription factor binding sites, for example UAS sites.  The Tet system is the same but you have a chemical modulator that conditions the transcription factor.

Two-component systems have an additional component that serves as a sensor (typically these are membrane proteins with histidine kinase activity, HK) that activates a transcription factor by phosphorylation (response regulator, RR) that in turn regulates the expression of a target gene.

Two-component systems are ubiquitous in prokaryotes with similar systems found in plants and some ‘lower’ eukaryotes. They are attractive for genetically engineering ‘higher’ eukaryotes (mammals, insects ?) because their ‘foreignness’ tends to minimize the risks of unwanted interactions. Or so the thinking goes.

Native 2-component system (A) and an engineered 2-component system (B). from Figure 1 in doi 10.1073/pnas.1406482111

Native 2-component system (A) and an engineered 2-component system (B).
from Figure 1 in doi 10.1073/pnas.1406482111

Hansen et al. look at three two-component signaling pathways EnvZ-OmpR, NarXL and DcuSR and introduce them into mammalian cells to evaluate their functionality.

Before using any of the components they were codon optimized for expression in mammalian cells without altering their core amino acid sequence however the RRs were modified to contain VP16 activating domains.

Hansen et al. find that all of these systems work when introduced into mammalian cells and expressed from mammalian promoters; in all cases the HK component was capable of phosphorylating the RR but in the absence of a ligand. This was a bit unexpected but it did not diminish the utility of the systems.

The authors demonstrate how these components, when combined with some ligand-regulated promoters and proteins, could be used to construct NOR and OR gated circuits.

The EnvZ/OmpR system is part of an osmoregulatory system with EnvZ being an osmosensor

The EnvZ/OmpR system is part of an osmoregulatory system with EnvZ being an osmosensor

Hansen et al. found that there was no cross talk from the host cell and there was very little, if any in some cases, between these three systems when they were introduced into the same cell.

This was an interesting technical paper that encourages one to think about circuit building in genetically modified insects. One is probably right to think that these two component systems would also work effectively in insects. Those experiments have yet to be done

 

Hansen J, Mailand E, Swaminathan KK, Schreiber J, Angelici B, Benenson Y (2014) Transplantation of prokaryotic two-component signaling pathways into mammalian cells. Proceedings of the National Academy of Sciences 111: 15705-15710 10.1073/pnas.1406482111

 

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