Team:Duesseldorf/Test4


Heinrich-Heine University
Universitätsstraße 1
40225 Düsseldorf
Design by Marvin van Aalst

Description

In Germany, every fourth death is caused by cancer. The malignant tumors are a threat to every organ in the body, however most are found in men’s prostate or mammary gland in women. The most deaths are caused by cancer located in breast and lungs. [1]

Every treatment in use has various side effects, which can affect the body more than the actual disease, weakening the sick person even more. A new cancer therapy without side effects is needed and can be realized using optogenetics due to its high accuracy. [2]

From the beginning, the goal of Optoptosis was to fight cancer and to ameliorate commonly known and used cancer therapies. Decreasing the patients’ suffering and improving their quality of living was our driving incentive. Treatments with state of the art cancer therapies are painful for the patients and they not only have to suffer from the disease, but also from the side effects of the therapy which may last up to a lifetime. The number of mortal cancer cases worldwide sums up to 8.2 million deaths per year. Resulting in one dead american citizen per minute. Current cancer therapies fail in stopping the mortality rate.

Reference: http://www.cdc.gov/cancer/international/images/worldwide-survivor-stackedchart.jpg

Our innovation has the potential to decrease the suffering through specifically targeting the lost function of self-induced cell death (apoptosis) in cancer cells. The process of apoptosis can be learned here.

Optoptosis uses two optogenetic proteins, namely LOV2 and Phytochrome B to combine the precision of light with the accuracy of viral vectors to induce apoptosis specifically in cancer cells. The red light switch controls expression through PhyB-VP16, while the blue light switch controls the localization of apoptotic proteins to their target site with the help of LOV2.

Mechanism of an optogenetic system for induction of apoptosis in cancer cell lines

The optogenetic induction of apoptosis in cell cultures (HeLa and CHO) serves as a model for the future application in situ. The application of optogenetic switches enables us to induct extremely precise and highly regulated elimination of malignant 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 the activation of apoptosis.

2. Spielerei mit den Lichtschaltern ON/OFF

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 (Q2) . Phycocyanobilin is ligated to the photosensory domain at the N-Terminus of PhyB which is, upon photoexcitation, responsible for conformational change. The activation through conformation change is responsible for binding to PIF6 (phytochrome interacting factor6). In response to red light (λ = 660nm) Phytochrome transits into its PhyBfr-conformation. In this state PhyB can interact with PIF6 through binding. [3]

[GRAFIK ARABIDOPSIS THALIANA] [sTRUKTURMODELL VON PHYCOCHROMOBILIN PHYCICYANOBILIN]

Q2: Phycocyanobilin, Phytochromobilin ( “The natural PhyB chromophore Phytochromobilin is not found in mammalian cells, but it can be substituted for PCB that is conveniently extracted from the cyanobacterium Spirulina and when added to the culture medium is autoligated to PhyB (9).” -”A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells “, Konrad Müller et. al p. 5: Funktionsweisen, woher kommt das (pflanzen, Tiere), wie wird das den Zellen zugeführt

The N-terminus of PIF6 is fused to tetR (tetracyclin Repressor), which constitutively binds the operator tetO upstream of a minimal promoter (Pmin). On the other hand, 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 (see fig. 1). [4]

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 protein BaxS184E lays in a fusion with the fluorescent protein mCherry and the Jα-binding PDZ-domain (see fig. 2). We use the hBax mutant BaxS184E, because it is not as deadly as the natural human BaxS184E protein. The BaxS184E mutant binds less effectively to the mitochondrial membrane and shows a decreased apoptotic potential in comparison to the non-mutated version. We plan to use this as an additional safety measure, so that the sole expression of BaxS184E does not cause apoptosis, but only if it is bound to the mitochondrial membrane with the help of the blue light switch construct. [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 human protein BaxS184E lays in a fusion with the fluorescent protein mCherry and the Jα-binding PDZ-domain. In our construct we used the weaker BaxS184E mutant BaxS184E. BaxS184E is not as deadly as the natural human BaxS184E because it is binding less effectively to the mitochondrial membrane and shows a decreased apoptotic potential. We planed to use this as an additional safety measure to prevent the BaxS184E from causing apoptosis by sole expression. Apoptosis only occurs when BaxS184E, triggered by our blue-light-switch construct is bound to the mitochondrial membrane.] We plan to use this as an additional safety measure, because the sole expression of BaxS148E does not cause apoptosis. Only when the blue light switch turns on the BaxS184E is bound to the mitochondrial membrane and the apoptosis is triggered. Quelle BaxS184e: https://www.ncbi.nlm.nih.gov/pubmed/20976235

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.

The fusion protein 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).

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]. Zitat aus (richtig zitieren) Component of the TOM (translocase of outer membrane) complex; responsible for recognition and initial import of all mitochondrially directed proteins; involved in transfer of precursors from the Tom70p and Tom20p receptors to the Tom40p pore 1 2

For our project, we used a double mutant of LOV2 derived from Avena sativa phototrophin 1 fused with a peptide epitope on the Jα-helix called AsLOV2pep (Tulip supplementary Fig. 2a and Supplementary Note 1). The peptide epitope consists of the following sequence –SSADTWV–COOH. AsLOV2pep is a double mutant. The original sequence of Jalpha is blub, the mutant sequence is blab. Abbildung Figure 2 aus sup8

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 from 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

https://static.igem.org/mediawiki/2016/a/a0/T--duesseldorf--01.png

Our AsLOV2pep is flanked with α-helices on the N- and C-terminals. Upon photoexcitation with blue light (λ = 472nm) the C-terminal Jα-helix from the AsLOV2-core and unfolds slightly (TULIP, Fig. 1b). It forms weak interactions with the α/β-scaffold of LOV2 .

mögliche antworten hier?: 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.

The exposure of the Jα-helix allows the interaction with a binding partner. The additional mutation of an epitope tag enables the Jα-helix to bind to ePDZ. ePDZ originally stems from mice, while LOV2 is derived from plants. using the associated peptide elongation for stronger binding (erst möglich zu binden, dadurch, dass pep an As LOV2 hängt kann ePDZ binden) [tulip]. It is now able to attach the ePDZ-domain of the other fusion protein, which contains BaxS184E. 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). Lovpep mutante mit ePDZb1 variante+ graphen aus paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766841/ ) hier?: "> Huang, J., Koide, A., Makabe, K. & Koide, S. Design of protein function leaps by directed domain interface evolution. Proc. Natl. Acad. Sci. USA 105, 6578–6583 (2008).

Here BaxS184E forms pores in the OMM allowing the release of cytochrome c, triggering 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, because a mutant form (Bax S184E) (Grund? oben) is used, which ability to localize to the OMM is lost. 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. verweis apoptose

References:

[1]= Peter Kaatsch, Claudia Spix, Alexander Katalinic, Stefan Hentschel, Nadia Baras, Benjamin Barnes, Joachim Bertz, Stefan Dahm, Jörg Haberland, Klaus Kraywinkel, Antje Laudi, Ute Wolf: Krebs in Deutschland 2007/2008 – Häufigkeiten und Trends. A publication from „Robert-Koch-Institut“ and „Gesellschaft der epidemiologischen Krebsregister in Deutschland e. V.“, 8. Edition, 2012, retrieved 3/15/2013
[2]= Enger, Eldon; et al. Concepts in Biology' 2007 Ed.2007 Edition. McGraw-Hill. p. 173. ISBN 978-0-07-126042-8. Retrieved 23 November 2012.
[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.
[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

[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
[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
evtl noch Quellen: 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. "> Strickland, D., Moffat, K. & Sosnick, T.R. Light-activated DNA binding in a designed allosteric protein. Proc. Natl. Acad. Sci. USA 105, 10709–10714 (2008).