Light on - Cancer gone

Mechanism of the Phytochrome-based expression system

The first optogenetic switch functions via Phytochrome B (PhyB) derived from Arabidopsis thaliana. PhyB’s natural chromophore is Phytochromobilin. Phytochromobilin is not found in mammalian cells but it is possible to use Phycocyanobilin extracted from Cyanobacteria instead ^[XY] . Phycocyanobilin is ligated to the photosensory domain at the N-Terminus of PhyB which is, upon photoexcitation, responsible for conformational change. In response to red light (λ = 660nm) Phytochrome transits into its PhyBfr-conformation and interacts with PIF6 (phytochrome interacting factor6) through binding, this rapid process takes 6.9 seconds. ^{[3] [XY3] [3.1]}

[STRUKTURMODELL VON PHYCOCHROMOBILIN PHYCoCYANOBILIN]

The N-terminus of PIF6 is fused to tetR (tetracyclin Repressor), which constitutively binds the operator tetO upstream of a minimal promoter (Pmin), while PhyB is fused to the transcription factor VP16. When the red light switch is activated, VP16 is recruited to the promoter region, so that 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 is terminated which takes 46.9 seconds ^[3.1] (see Buckley et al. (2016) fig. 1). ^[4]

Figure 1: The phytochrome-based expression system

The PDZ-mCherry-BaxS184E construct, which expression is regulated by the PhyB-based switch, represents a component of the second optogenetic switch that is based on LOV2. The BaxS184E lays in a fusion with the fluorescent protein mCherry and the Jα-binding PDZ-domain. In our construct we used the weaker Bax mutant BaxS184E ^[XY2] because apoptosis only occurs when BaxS184E, triggered by our blue-light-switch construct, is bound to the mitochondrial membrane.

apoptosis[Why do we use BaxS184E? Link]

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

---

Expression of fusion proteins utilizing a constitutive promoter

Another construct needed for the LOV2-based optogenetic switch is expressed constitutively in the cells. For this purpose, expression of this construct is brought under control of the pSV40 viral promoter.

Our second, blue-light switch is a fusion protein and consists of the mitochondrial anchor TOM5 (translocase of the outer membrane 5), 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: Constitutive expression system for the expression of the LOV2 -based optogenetic switch?

TOM5 is a mitochondrial protein that is responsible for recognizing and initially importing of all proteins directed to the mitochondria. Moreover, it is involved in transfer of precursors from the Tom70p and Tom20p receptors to the Tom40p pore, which are supposedly responsible for porin import into the mitochondria ^{[6] [7]}.

LOV2 is a protein sensor domain, which function is photosensing in natural organisms such as Avena sativa. An important structure of the domain is the anchor Jα-helix. In our approach, we use the LOV2 domain to control the localization of the apoptotic construct to the outer mitochondrial membrane. More precisely, the structure of the double-mutated version LOV2pep allows the binding of the ePDZ domain (see fig. 2).
[For more information on what our LOV2pep makes special, click here.]

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

Once both components of the LOV2-switch have been synthesized and brought automatically to their target site they are ready to interact. In order to absorb light, the LOV2 protein needs the chromophore FMN which is produced by the cells themselves and binds to the α/β-scaffold of LOV2. The inactivated state of LOV2 is called D450 and converts to the activated State S390 after blue-light induction

Our LOV2 is flanked with α-helices on the N- and C-terminals. Upon photoexcitation with blue light (λ = 473nm) the C-terminal Jα-helix from the LOV2-core undocks and unfolds slightly (see TULIP Fig. 1b). It forms weak interactions with the α/β-scaffold of LOV2

[Grafik Tulip Fig. 1b]

The exposure of the Jα-helix allows the interaction with a binding partner. The additional mutation of an peptide epitope tag enables the Jα-helix to bind to ePDZ. ePDZ originally originates from mice, while LOV2 is derived from Avena sativa ^[8] [For more information on ePDZ click here]. It is now able to attach the ePDZ-domain of the other fusion protein, which contains BaxS184E (see fig. 2). LOV2 is bound to the OMM (outer mitochondrial membrane) due to its mitochondrial anchor TOM5. Therefore, binding between Jα and ePDZ causes recruitation of BaxS184E to the OMM (fig. 4).

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

Here BaxS184E forms pores in the OMM allowing the release of cytochrome c, inducing apoptosis (fig. 5). So BaxS184E 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 BaxS184E to the mitochondria does not occur. Thus BaxS184E 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.

---

GLOSSAR:

Why do we use BaxS184E?

One natural apoptotic protein in humans is called hBax. Its ability to induce apoptosis is very strong and the constitutive expression of hBax results in definite death of the cell. Since OPTOPTOSIS should be applied as a therapy in humans in the future, we searched for a suitable substitution and found one.

BaxS184E is not as deadly as the natural human Bax because it is binding less effectively to the mitochondrial membrane due to a substitution of serine to glutamate in the residue S184 in the C-terminus of Bax. Due to the substitution, the previous phosphorylation at S184 is malfunctioning resulting in conformational changes. Because of those changes Bax’s proapoptotic function may be inactivated or at least decreased, since the phosphorylation at S184 is important for the cytosolic retention of Bax. Plus, an autonomous localization of BaxS184E to the mitochondria does not occur, because its ability to localize to the OMM is lost. To induce apoptosis BaxS184E has to be brought in vicinity to the outer mitochondrial membrane. ^[XY2]

because a mutant form (Bax S184E) (Grund? oben) https://static.igem.org/mediawiki/2016/a/ac/T--duesseldorf--baxexpression.png

Figure 1: Expression of various mutations of Bax, transfection into Bax−/− MEF cells and Western-Blot of viable cells in comparison to vector-only control to measure the function and potency of mono-or double-site Bax phosphorylation and function of conformational changes.^[XY2]

What makes our LOV2 special? AsLOV2pep mutant

For the localization of our apoptosis product we needed a protein that was photo-inducible and regulatable. Plants metabolistic actions are dependent on light energy and have evolved diverse light receptors, eg. phototrophin which was extensively researched for optogenetic purposes. Phototrophin has two LOV domains, called LOV1 and LOV2. Each domain is responsible for binding one chromophore FMN (Flavo mononucleotide), so that light energy can be accepted.

For our project, we used the double mutant version of LOV2 derived from Avena sativa’s phototrophin 1 fused with a peptide epitope on the Jα-helix called AsLOV2pep. We used this mutant form of LOV2, because of the fact that it has a higher dark binding affinity to the Jα-helix (150µM instead of 72µM). The higher affinity is caused by the substitutions at T406-7AI532. Through these changes the possibility that the Jα-helix epitope is exposed during dark state, is greatly reduced ^{[8] [XY8]}. (Tulip supplementary Fig. 2a and Supplementary Note 1).

[More details about AsLOV2pep here LINK]

Chem Biol. 2012 Apr 20;19(4):507-17. doi: 10.1016/j.chembiol.2012.02.006. Designing photoswitchable peptides using the AsLOV2 domain.Lungu OI1, Hallett RA, Choi EJ, Aiken MJ, Hahn KM, Kuhlman B.

---

ePDZ-b1 attracted to AsLOV2pep

ePDZ-b1 is used in our construct, this pdz domain is engineered to have a higher affinity to the bounding peptide. The binding kinetics were measured and resulted into a 500 times higher affinity =60nM. ^[XY11] The structure of the Erbin PDZ bound to a peptide (PDB entry 1MFG). The N and C termini are indicated. The positions for the new termini of the circularly permutated PDZ (cpPDZ) are shown with a triangle and residue numbers. Right shows the surface of the PDZ domain with the peptide as a stick model, illustrating the shallow binding pocket. ^[XY11] Figure 1.C A cycle of affinity maturation of ePDZ-b produced second-generation affinity clamps with Kd values in the single-nanomolar range and dissociation half-lives of nearly 1 h (termed ePDZ-b1 and ePDZ-b2, respectively; Fig. 2B and Table 1). These values are comparable to those found for antibody–antigen interactions. Importantly, the affinity enhancement of >6,000-fold relative to cpPDZ (>500-fold relative to wild-type PDZ) (Table 1) by the affinity clamp strategy is far superior to the enhancement achieved by simple optimization of the peptide-binding interface of another PDZ domain alone (25), demonstrating the capacity of directed domain interface evolution to acquire function that is otherwise unattainable by manipulating only the primary domain.^[XY11] They bind either the carboxyl-terminal sequences of proteins or internal peptide sequences [PMID: 9204764]. In most cases, interaction between a PDZ domain and its target is constitutive, with a binding affinity of 1 to 10 microns. ^[XY9]

---

Apoptosis

Apoptosis is an active process of cell death, which is very important for homeostasis and development of metazoan organisms. For instance it is necessary for the regeneration of the intestinal epithelium, the metamorphosis of frogs and the formation of toes and fingers in embryogenesis in mammals.

In opposite to necrosis, it is a regulated process which neither results from an acute cell injury nor does it lead to an inflammation of tissue, because of leaking cell content. The process of apoptosis is controlled by enzymes, called caspases. In the course of the process, cell fragments called apoptotic bodies were produced. They contain fragmented DNA and other cell organelles. Furthermore, the membrane lipid phosphatidylserine, which is normally localized in the inner monolayer and vital for intracellular transduction pathways or exocytosis, shows up in the outer monolayer of the lipid bilayer. Therefore macrophages are able to identify the apoptotic bodies by the phosphatidylserine, whereby the apoptotic bodieS are absorbed and digested by the macrophages. ^[A]

There are two different ways to induce apoptosis. The first one is the extrinsic pathway, triggered through extracellular stimuli like a Fas ligand on a killer lymphocyte, which links to a Fas receptor on the target cell. This leads to activation of Caspase 8 and 10 and finally to apoptosis. ^[B] The second way to induce apoptosis is the intrinsic pathway. For instance this pathway is triggered through DNA damage. Finally, cytochrome c, which normally is content of the cellular respiration only within the mitochondrion, streams out and leads to apoptosis. ^[C]

Our project focuses on the intrinsic pathway, more precisely on the outflow of cytochrome c at the outer mitochondrial membrane (OMM), which is the consequence of the activation of proteins from Bcl-2 family. Proapoptotic proteins from this family, like Bax and Bak, are able to form channels in lipid membranes through heterodimerization, for example in the outer mitochondrial membrane, which leads to permeability transition. Cytochrome c is able to pass through the pores in the outer mitochondrial membrane.

The Cytochrome c activates a cytosolic protein called Apaf-1, which has an N-terminal CAR domain (caspase recruitment domain). This CAR domain induces the self-cleavage/activation of caspase -9 through heterodimerization. This complex is called apoptosom and leads to activation of more caspases which induce apoptosis. ^[D]

Relating to our project, the apoptotic proteins were produced inside the cell through the optogenetic protein phytochrome b which is activated by red light. Another requirement for outstream of cytochrome c is the spatial proximity of the Bax or Bak protein at the outer mitochondrial membrane for increasing the permeability transition. Therefore the second optogenetic protein called LOV (light oxygen voltage) is activated through blue light and localizes the apoptotic protein at the mitochondrion.

---

References :

^[XY] Müller K., Zurbriggen MD., Weber W. (2014), Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch. Nature Protocols 9, pp 622-632. doi:10.1038/nprot.2014.038
^[3]= Khanna,R., Huq,E., Kikis,E.A., Al-Sady,B., Lanzatella,C. and Quail,P.H. (2004) A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. Plant Cell, 16, 3033–3044.
^[3.1] Buckley CE, Moore RE, Reade A, Goldberg AR, Weiner OD, Clarke JDW (2016). Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo. Dev Cell 36(1), pp 117-126. doi:10.1016/j.devcel.2015.12.011
^[XY3] Levskaya,A., Weiner,O.D., Lim,W.A. and Voigt,C.A. (2009), Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 461, 997–1001.
^[4]= Müller et al.(2013) A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells, Nucleic Acids Research, 2013, Vol. 41, No. 7 e77 doi:10.1093/nar/gkt002
^[XY2] Wang Q., Sun SY, Khuro F, Curran WJ, Deng X (2010). Mono- or double-site phosphorylation distinctly regulates the proapoptotic function of Bax. PLos One, p .doi: 10.1371/journal.pone.0013393.
^[6]= Krimmer T., Rapaport D., Ryan Michael T., Meisinger C., Kenneth Kassenbrock C., Blachly-Dyson E., Forte M., Douglas Michael G.Neupert W., Nargang Frank E., Pfanner N. (2001 Jan. 22), Biogenesis of Porin of the Outer Mitochondrial Membrane Involves an Import Pathway via Receptors and the General Import Pore of the Tom Complex, J Cell Biol., Vol. 152(2): 289–300. PMCID: PMC2199606
^[7]= http://www.yeastgenome.org/locus/tom5/overview [last access: 10/16/2016]
^[8] = Strickland D., Lin Y., Wagner E., Hope M., Zayner J., Antonious C., Sosnick T.R., Weiss E.L., Glotzer M. (2012), TULIPS: tunable, light-controlled interacting protein tags for cell biology, Nature Vol.9(4), doi:10.1038
^[XY8] Hallet RA, Zimmermann SP, Yumerefendi H, Bear JE, Kuhlmann B (2016). Correlating in vitro and in vivo Activities of Light Inducible Dimers: a Cellular Optogenetics Guide. ACS Synth Biol. 5(1), pp 53-64. doi: 10.1021/acssynbio.5b00119
^[XY9] Ponting CP, Phillips C, Davies KE, Blake DJ (1997). BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology. University of Oxford, Fibrinolysis Research Unit, UK. Vol19(6):469-479]. doi: 10.1002/bies.950190606 ^[9] =Okajima K. (2016), Molecular mechanism of phototropin light signaling, J Plant Res 129(2):149-157. doi: 10.1007/s10265-016-0783-6
^[10] = Halavaty AS, Moffat K (2007), N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototrophin 1 from Avena Sativa. Biochemistry 46:14001-14009 ^[XY11] Huang J, Koide A, Makabe K, Koide S (2008). Design of protein function leaps by directed domain interface evolution. Proc Natl Acad Sci USA 105, pp 6578-6583. doi: 10.1073/pnas.0801097105 ^[A]=J Biol Chem. 1997 Oct 17;272(42):26159-65. Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase. Bratton DL1, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM. ^[B]=FEBS Letters 1995-10-16 Interaction of peptides derived from the Fas ligand with the Fyn-SH3 domain. M Hane, B Lowin, M Peitsch, K Becker, J Tschopp ^[C]=Annu Rev Biochem. 2004;73:87-106. Cytochrome C-mediated apoptosis. Jiang X1, Wang X. ^[D]=Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? November 1998 Yoshihide Tsujimoto