Engineering Signal Transduction Pathways Christina Kiel, Eva Yus, Luis Serrano  Cell  Volume 140, Issue 1, Pages 33-47 (January 2010) DOI: 10.1016/j.cell.2009.12.028 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 The Engineering of Signal Transduction Pathways and Gene Networks When engineering signal transduction pathways as opposed to gene circuits, there are two main points to consider: the general properties of the system and its components. Regarding the components, nucleotides can usually be exchanged without having a large impact on neighbor nucleotides and DNA structure (“independence approximation”). In contrast, amino acid substitutions in proteins can lead to structural changes and misfolding. With respect to the circuit properties, gene circuits are modular, they usually respond slowly, and the responses can be stochastic. In contrast, signaling pathways are highy modular, respond fast, often involve signal amplication, and make use of spatial localization. Cell 2010 140, 33-47DOI: (10.1016/j.cell.2009.12.028) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Signaling Pathway Engineering in Prokaryotes and Eukaryotes Signal transduction can be engineered on three different levels, illustrated here using three examples: (1) pathway modulation, for example by protein mutagenesis, (2) the rewiring of existing pathways, which can be achieved by creating chimeric proteins that have new input-output domains, and (3) the creation of artificial signal transduction pathways and cells. Cell 2010 140, 33-47DOI: (10.1016/j.cell.2009.12.028) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Modulation of Signal Transduction (A) A Ras mutant (V12, G37) that is unable to activate the effector protein c-Raf has been used to study c-Raf-independent effector pathways in human T cells (Czyzyk et al., 2003). (B) When human growth hormone (GH) is modified with a G120R mutation, it induces conformational changes in the growth hormone receptor (GHR) such that it can activate the Jak/STAT pathway but not the ERK pathway (Rowlinson et al., 2009). (C) Using computational design, a TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mutant has been created such that it can only bind to the DR5 receptor, and not to TRAIL's other receptors. This specificity induces faster kinetics of receptor activation and induction of apoptosis in colon carcinoma (Colo205) cells (E. Szegezdi, L.S., and A. Samali, unpublished data). (D) Using structure-based design, various c-Raf mutants have been made that alter the kinetics of its association with Ras. The effect on signal transduction of these mutants strongly depends on the network topology: the effect is minor under conditions of strong negative feedback in human embryonic kidney cells (HEK293), but a strong effect is observed in a cell line with reduced negative feedback (rabbit kidney cells, RK13) (Kiel and Serrano, 2009). (E) A system for controllable degradation of proteins has been devised in the yeast Saccharomyces cerevisiae that involves the expression of a modified ClpXp protease from the bacterium Escherichia coli. Target proteins are expressed with a ssrA tag, which is specifically recognized by the ClpXp proteasome (Grilly et al., 2007). EGFP, enhanced green fluorescent protein. (F) A system for controllable protein degradation in E. coli also uses the ClpXp system and target proteins tagged to ssrA. For better control of degradation, an additional feature is the inclusion of the adaptor protein SspB, which increases the efficiency of degradation, is expressed under an inducible promoter (McGinness et al., 2006). Cell 2010 140, 33-47DOI: (10.1016/j.cell.2009.12.028) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Rewiring of Signal Transduction Pathways (A) Combining different guanine nucleotide exchange factor (GEF) domains with an artificial regulatory motif has been used to rewire GTPase signaling. The regulatory motif consists of a PDZ domain and a PDZ binding motif, rendering the GEF inactive by autoinhibition. Activation is achieved by cAMP induction, PKA activation, and phosphorylation of the PDZ binding motif (Yeh et al., 2007). (B) In RAT-2 rat fibroblasts epidermal growth factor (EGF) signaling has been rewired by fusing SH2 or PTB domains of the adaptor proteins Grb2 and Shc to the dead domain (DED) of FADD. Consequently, cell death is triggered after stimulation by EGF (Howard et al., 2003). (C) A chimera between the light-sensing part of the G protein coupled receptor (GPCR) of Rhodopsin and two other GPCRs, the β2 and α1 adrenergic receptors, results in light stimulated β2 and α1 specific cellular responses in human embryonic kidney (HEK293) cells and in adult mice (Airan et al., 2009). (D) Light-dependent activation in the bacterium Escherichia coli is achieved by a chimera of a cyanobacterial photoreceptor (PCB) with the EnvZ-OmpR two-component system. The reporter is modified to produce a black compound, so E. coli cells when grown on agar, produce a light dependent image (“bacterial photography”) (Levskaya et al., 2005). (E) Saccharomyces cerevisiae cells were engineered for cell-to-cell communication with elements from the model plant Arabidopsis thaliana. Sender cells produced a plant cytokinin (IP), which could diffuse to receiver cells and bind to the plant receptor AtCRE1, and consequently induced the yeast YPD1 signaling cascade (Chen and Weiss, 2005). (F) A cell-to-cell communication system was engineered in the marine bacterium Vibrio fischeri, which produced a diffusible acyl-homoserine lactone (AHL). Upon a critical cell density, AHL together with a transcriptional regulator (LuxR) induced the production of a “killer gene” (E) (You et al., 2004). Cell 2010 140, 33-47DOI: (10.1016/j.cell.2009.12.028) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Artificial Signal Transduction and Cells (A) Introduction of a minimal human p53 signaling module into yeast, as a model system to study context independent human p53-Mdm2 interactions (Di Ventura et al., 2008). (B) In the bacterium Escherichia coli, partial artificial modules from the mevalonate pathway of the yeast Saccharomyces cerevisiae have been engineered to produce a terpenoid precursor for artemisinin, an antimalarial drug (Martin et al., 2003). (C) In the model plant Arabidopsis thaliana, a cytokinin response has been coupled to an E. coli two-component system (PhoB and OmpR) (Antunes et al., 2009). GFP, green fluorescent protein. (D) Transplantation of the genome of Mycoplasma mycoides into M. capricolum, via cloning of the M. mycoides genome as a yeast centromeric plasmid. This opens the possibility of engineering a bacterial genome in yeast, before transplanting in into the host bacterium (Lartigue et al., 2009). Cell 2010 140, 33-47DOI: (10.1016/j.cell.2009.12.028) Copyright © 2010 Elsevier Inc. Terms and Conditions