Tunable Signal Processing in Synthetic MAP Kinase Cascades

O’Shaughnessy, Ellen C.; Palani, Santhosh; Collins, James J.; Sarkar, Casim A.

doi:10.1016/j.cell.2010.12.014

Cited by 139 publications

(146 citation statements)

References 54 publications

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“…In recent years synthetic biology has greatly advanced the design of biomedical devices (19)(20)(21)(22) that provide new diagnostic tools (23); offer novel concepts for the treatment of cancer (24,25), immune diseases (26,27), and diabetes (28); and pioneered prosthetic networks as a therapy for hyperuricemic disorders such as the tumor lysis syndrome and gout (29). Still, all synthetic biology-based treatment strategies target a single disorder at a time, taking advantage of a specific therapeutic nucleic acid or protein compound.…”

Section: Discussionmentioning

confidence: 99%

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Hamri

Zwicky

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic biology has significantly advanced the design of genetic devices that can reprogram cellular activities and provide novel treatment strategies for future gene-and cell-based therapies. However, many metabolic disorders are functionally linked while developing distinct diseases that are difficult to treat using a classic one-drug-one-disease intervention scheme. For example, hypertension, hyperglycemia, obesity, and dyslipidemia are interdependent pathologies that are collectively known as the metabolic syndrome, the prime epidemic of the 21st century. We have designed a unique therapeutic strategy in which the clinically licensed antihypertensive drug guanabenz (Wytensin) activates a synthetic signal cascade that stimulates the secretion of metabolically active peptides GLP-1 and leptin. Therefore, the signal transduction of a chimeric traceamine-associated receptor 1 (cTAAR1) was functionally rewired via cAMP and cAMP-dependent phosphokinase A (PKA)-mediated activation of the cAMP-response element binding protein (CREB1) to transcription of synthetic promoters containing CREB1-specific cAMP response elements. Based on this designer signaling cascade, it was possible to use guanabenz to dose-dependently control expression of GLP-1-Fc mIgG -Leptin, a bifunctional therapeutic peptide hormone that combines the glucagon-like peptide 1 (GLP-1) and leptin via an IgG-Fc linker. In mice developing symptoms of the metabolic syndrome, this three-in-one treatment strategy was able to simultaneously attenuate hypertension and hyperglycemia as well as obesity and dyslipidemia. Using a clinically licensed drug to coordinate expression of therapeutic transgenes combines drug-and genebased therapies for coordinated treatment of functionally related metabolic disorders. T he metabolic syndrome is a combination of disorders and risk factors including hypertension, hyperglycemia, obesity, and dyslipidemia that show an extremely complex and littleunderstood interdependent pathophysiology and collectively increase the risk for cardiovascular diseases that remain the prime cause of mortality worldwide (1-8). Currently available therapies independently target each of the individual disorders and risk factors, but a well-coordinated collective treatment strategy does not exist (2). We have therefore designed a synthetic signaling cascade in which a clinically licensed small-molecule drug finetunes expression of therapeutic transgenes. The combination of drug-and gene-based therapies enables simultaneous treatment of all key metabolic syndrome pathologies. Guanabenz (Wytensin) is a clinically licensed antihypertensive drug that is thought to activate alpha-2-selective adrenergic receptors in the central nervous system, thereby reducing the sympathetic outflow to heart, kidney, and peripheral vasculature and decreasing the systolic and diastolic blood pressure (9). Recently, guanabenz was also identified as an agonist of the trace amine-associated receptor 1 (TAAR1) (10). TAARs are G protein-coupled receptors located in the neu...

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Section: Discussionmentioning

confidence: 99%

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Hamri

Zwicky

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The S. pombe pheromone MAPK module, like the mammalian Raf-MEK-ERK cascade, which can functionally replace it (Hughes et al 1993), lacks a scaffold protein. Interestingly, synthetic biological approaches have shown that a simple concentration increase of sequential Raf-MEK-ERK kinases is sufficient to lower the activation threshold and enhance ultrasensitivity, leading to full activation (O'Shaughnessy et al 2011). This evidence leads us to propose that the local concentration of the pheromone MAPK cascade at the fusion site in S. pombe represents a natural situation in which the cell exploits the ultrasensitivity of the MAPK cascade to convert it to the fully active state, which, due to the spatially restricted location in which this takes place, drives an essentially irreversible decision to fuse.…”

Section: Spatial Focalization Of Mapk Signaling As a Cellular Decisionmentioning

confidence: 99%

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell–cell fusion

2016

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Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone-GPCR (G-protein-coupled receptor)-MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell-cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception.

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“…U nderstanding the information processing abilities of biological systems is a central problem for systems and synthetic biology (1)(2)(3)(4)(5)(6). The properties of a living system are often inferred from the observation of its response to static perturbations.…”

mentioning

confidence: 99%

Long-term model predictive control of gene expression at the population and single-cell levels

Uhlendorf

Miermont

Delaveau

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

220

237

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Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells' environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.model based control | computational biology | high osmolarity glycerol pathway | quantitative systems biology U nderstanding the information processing abilities of biological systems is a central problem for systems and synthetic biology (1-6). The properties of a living system are often inferred from the observation of its response to static perturbations. Timevarying perturbations have the potential to be much more informative regarding the dynamics of cellular functions (7-12). Currently, it is not possible to precisely perturb protein levels in an analogous manner, even though this perturbation would be instrumental in our understanding of gene regulatory networks. Indeed, despite the development of novel regulatory systems, including various RNA-based solutions (13), transcriptional control by means of inducible promoters is still the preferred method for manipulating protein levels (14, 15). Unfortunately, inducible promoters have several generic limitations. First, there is a significant delay between gene expression activation and effective protein synthesis. Second, many cellular processes can interfere with gene expression through internal feedback loops whose effects are hard to predict. Third, the process of gene expression shows significant levels of noise (16)(17)(18). Given these limitations, novel experimental strategies are required to gain quantitative, real-time control of gene expression in vivo.Here, we see the...

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Tunable Signal Processing in Synthetic MAP Kinase Cascades

Cited by 139 publications

References 54 publications

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell–cell fusion

Long-term model predictive control of gene expression at the population and single-cell levels

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