Team:Duesseldorf/Description

Mechanism of an optogenetic system for induction of apoptosis in Saccharomyces cerevisiae

The optogenetic induction of apoptosis in the budding yeast (S. cerevisiae) should serve us as a model for the future application in cancer cells. The application of optogenetic switches enables us to induct extremely precise and multiply regulated killing of cells. Thereby this system will represent an improvement in comparison to conventional, less target-specific methods. The sequential utilization of two optogenetic switches, namely a phytochrome-based expression system and a LOV2-based switch needed for the localization of apoptotic proteins to the outer mitochondrial membrane, allows the attainment of a very high level of spatiotemporal specificity for apoptotic activation.

Mechanism of the phytochrome-based expression system

The first optogenetic switch functions via phytochrome B (phyB) derived from Arabidopsis thaliana. In response to red light (λ = 660nm) phytochrome transitions into its phyBfr-conformation. In this state phyB can interact with PIF6 (phytochrome interacting factor 6). PIF6 is fused to tetR, which constitutively binds the operator tetO upstream of a minimal promoter (Pmin). On the other hand phyB is fused to the transcription factor VP16, which is recruited to the promoter region, when the switch is activated by red light. The vicinity of VP16 to the promoter region allows initiation of transcription. Far-red light (λ = 740nm) is applied to the system in order to deactivate the switch. Under far-red light phyB reverts back to its phyBr-state and interaction with PIF6 and hence initiation of transcription can no longer occur (see fig. 1).

Figure 1: The phytochrome-based expression system

The first construct, which expression is regulated by the phyB-based switch, represents a component of the second optogenetic switch (based on LOV2). The human apoptotic protein hBAX is fused to the fluorescent protein mCherry and the Jα-binding PDZ-domain (see fig. 2).

Figure 2: Expression of a component of the LOV2-based optogenetic switch

Expression of fusion proteins utilizing the GAL1-expression system

Another construct needed for the LOV2-based optogenetic switch is only expressed after induction with galactose. For this purpose expression of this construct is brought under control of the galactose-inducible GAL1-expression system. The fusion protein consists of the mitochondrial anchor TOM20 (translocase of the outer membrane 20), the fluorescent protein GFP (green fluorescent protein) and the optogenetic protein LOV2 (light-oxygen-voltage-sensing 2) derived from Avena sativa (see fig. 3). The C-terminus of LOV2 contains the so called Jα-helix (see fig. 3), which allows binding with PDZ (see fig. 2).

Figure 3: GAL1-expression system for the expression of the LOV2-based optogenetic switch

The LOV2-based optogenetic switch allows localization of apoptotic proteins the outer mitochondrial membrane

Once both components of the LOV2-switch have been synthesized and brought to their target site, they can interact. Exposing LOV2 to blue light (λ = 472nm) changes its conformation with the consequence that the C-terminal Jα-helix is set free and can interact with a binding partner. Jα is now able to bind the PDZ-domain of the other fusion protein, which contains hBAX. LOV2 is bound to the OMM (outer mitochondrial membrane) due to its mitochondrial anchor TOM20. Therefore binding between Jα and PDZ causes recruitment of hBAX to the OMM (fig. 4).

Figure 4: The LOV2-based optogenetic switch is activated by blue light.

Here hBAX forms pores in the OMM allowing the release of cytochrome c, triggering apoptosis (fig. 5). So hBAX is only capable of fulfilling its function, when its expression has firstly been activated by the phyB-based switch and secondly, when it has been recruited to the mitochondria by activation of the LOV2-based switch. An autonomous localization of hBAX to the mitochondria does not occur, because a mutant form (hBAX S184E) is used, which ability to localize to the OMM is lost. Thus hBAX will only be found at its target site after activation of the blue light regulated switch. The fluorescent proteins GFP and mCherry serve as markers.

Figure 5: Release of cytochrome c from the mitochondrial intermembrane space triggers apoptosis.

Future application in cancer cells

In contrast to human cells, cancer cells have lost the ability to recognize their own devastating impact on the organism. Thus they do not induce apoptosis, but rather show uncontrolled growth leading to severe consequences for health. With our optogenetic system we intend to reestablish the natural balance between life and death, proliferation and apoptosis, in impaired tissue. The future objective of this research is to implement our system in tumors via virotherapy. Therefore our idea represents the perfect complement to the already innovative virotherapy.