5. Stand der Wissenschaft und Technik, bisherige eigene Arbeiten, Patentlage,
Wirtschaftliche Bedeutung
A. Title of the project
System biological analysis of rebuild signaling pathways
B. Short Summary
Many intracellular signaling elements have been identified in recent years, but their
spatial and temporal relationship with each other and with their receptor are poorly
understood. Furthermore for any mathematical modeling it is important to obtain also
kinetic and quantitative of signaling processes. We have developed a method
allowing the reconstitution of mammalian receptors and their signaling cascade in the
genetically distant environment of a Drosophila S2 Schneider cell. This synthetic
biology approach allows us to freely choose and alter the components of a signaling
pathway. Furthermore by controlling the protein expression and function in an
inducible fashion we can modify the kinetic and quantitative parameters of signaling
pathways. This new approach will be used to verify or falsify mathematical models of
signaling.
C. Specific Aims
Inside the cells signals are processed by a multitude of proteins each of which has its
own unique structural and regulatory features as well as different kinetics parameters
for signaling. To cope with this complexity, there exist many approaches to generate
mathematical models of signaling networks. However, a model is only as good as it
can be used to make clear predictions about the outcome of a signaling process
inside the cell. To address these aspects of signaling a synthetic-biological approach,
namely the rebuilding of signaling pathways can be an important new tool for a better
qualitative and quantitative analysis of signaling pathways. By rebuilding a given
signaling pathways the experimenter is free to either omit or alter every component of
a signaling system. Furthermore by employing inducible techniques for gene
expression the experimenter cannot only change the functional but also the
quantitative aspect of a signaling network. As most cellular signaling processes
require a membrane and a normal cellular environment for their proper function, such
a signal reconstitution cannot be done in a test tube. One solution to this problem is
the rebuilding of a mammalian signaling pathway in a cell that lacks most
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components of this pathway. The Drosophila S2 Schneider cells are fulfilling these
requirements (Towers and Sattelle, 2002). Indeed, these cells can be efficiently
cotransfected in a transient manner with up to 15 plasmids each of which carrying a
different gene of a signaling pathway (Rolli et al., 2002). The transient transfection
method used in the S2 reconstitution system allows to analyze in a short time frame
many mutants of given signaling proteins. Another advantage of the S2 cell system
is, that any Drosophila protein that may interfere with the rebuilt signaling pathway,
once identified, can efficiently be eliminated by the RNAi technique, which works very
efficiently in S2 cells (Caplen et al., 2000). Furthermore each plasmid carries a
metallothionein promotor that regulates the expression of the transient transfected
gene in an inducible fashion. This allows to study the behavior of a signaling network
at different levels of protein production and thus analyse the quantitative aspects of
signaling. At present, our experimental read-outs in this system are kinase activation,
substrate phosphorylation, protein-protein interaction and signaling complex
assembly (Wossning and Reth, 2004).
To cope with the complexity of signaling networks, there exist many approaches to
generate mathematical models of these signaling. We will apply our S2 Schneider
cell Drosophila system to rebuild mammalian signaling pathways, from which
mathematical models have been generated and use this system to verify or falsify
these theoretical models. The rebuilding approach allows us to omit or change by
mutation any component of a given signaling pathway. Furthermore, the inducible
fashion, with which we activate gene expression in the S2 cell system allows us not
only to change the components, but also the amount of signaling elements in this
system. However, right now, in the S2 cells we are only applying regulated promotor
for the inducible expression of signaling components. To better study not only the
quantitative aspect, but also the kinetic aspect of a signaling pathway it would be
important to also have the possibility to induce signaling pathways at a short time
frame (in seconds and minutes). To achieve this we will express fusion proteins
between the hormone-binding domain of a mutant and Tamoxifen inducible estrogen
receptor (ERT2) and kinase domains, which are critical activators of certain signaling
pathways. Such ERT2 kinase fusion proteins can be activated in a very short time
frame and allow to critically test kinetic parameters of signaling models (Verrou et al.,
1999).
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D. Background and Significance
Reverse engineering of intracellular signaling pathways.
Crucial cell fate decisions like proliferation, differentiation, survival or death are
regulated by a multitude of extra- and intracellular signals. Extracellular signals, such
as growth factors are sensed by membrane-bound receptors that translate the extracellular signal into an intracellular activity. Inside the cells signals are processed
through multiple pathways resulting in their amplification and regulation by critical
feedbacks. The outcome of these signal processes are defined changes in the
metabolism, the cytoskeleton and the movement as well as in the transcriptional
program of the cell. Due to the concerted efforts of genomic and proteomic
approaches many of the components of intracellular signaling pathways have been
identified in the last years. However, little knowledge exists about the mechanistic
and quantitative aspect of intracellular signaling processes. In Freiburg several
research groups have a longstanding interest in studying signaling pathways by
either genetic or biochemical means (See study of the insulin receptor pathway,
Baumeister group, XX, YY, Signal transduction from the B cell antigen receptor, Reth
group). Furthermore, there exists active collaboration with bioinformatic and
theoretical groups to generate mathematical models of these signaling pathways
(see Timmer group). At present most signaling studies by genetic means are done by
the generation of loss of function mutants via the RNAi or knock-out technology.
These approaches are very important to identify the functional importance of a given
signaling element or a given signaling pathway. However, the mechanistic and the
qualitative aspect of signaling pathways as well as the critical feedback loops, which
critically determine their outcome, are hard to study by these loss-of-function
approaches alone. To address these aspects of signaling a synthetic-biological and
reverse engineering approach, namely the rebuilding of signaling pathways from
known components can be a powerful research tool for a better and qualitative
analysis of signaling pathways (Arkin, 2001; Blake and Isaacs, 2004; Guet et al.,
2002; Pawson and Linding, 2005). Indeed the integration of these engineering
concepts with computational methods can lead us to a deeper and more abstract
understanding of signal transduction systems (Simpson, 2004).
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Guet, C. C., Elowitz, M. B., Hsing, W., and Leibler, S. (2002). Combinatorial
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Rolli, V., Gallwitz, M., Wossning, T., Flemming, A., Schamel, W. W., Zurn, C., and
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