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AMPK promotes S-phase entrance by controlling CLB5 transcription in budding yeast
S520
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
cose synthetic media, deletion of the SNF1 gene yields a severe slow growth phenotype and a specific delay in the execution of the G1 to S transition, while the expression of the non-phosphorylatable mutant protein Snf1-T210A gave only a partial complementation of the growth rate. Thus we provided a previously unrecognized role for Snf1 in regulating the S-phase entrance, the area of major relevance for cell cycle control in budding yeast, and we showed that this role is partially independent from Snf1 activation. Our experimental results reported above are part of a modular systems biology approach, developped by our group, that relies on molecular analysis of perturbed cell cycle for regulatory interaction elucidation and networks identification in order to construct a comprehensive mathematical model of the yeast cell cycle. doi:10.1016/j.jbiotec.2010.09.832 Fig. 3.
[P-S.26] tems), DPP, tau-DPP, Stau-DPP. The software supports SBML format, and can automatically convert stochastic models into the corresponding deterministic formulation. Deterministic simulations can be performed by using different ODE solvers. Parameter estimation is executed through optimization methods as Particle Swarm Optimizer, Genetic Algorithms and Hill Climbing. Results: BioSimWare has been exploited for the investigation of many biological systems, such as the Ras/cAMP/PKA pathway in yeast, bacterial chemotaxis, a synthetic genetic oscillator coupled with quorum sensing, metapopulations, etc. (papers published in international journals). Discussion: The novelty of BioSimWare is that it allows the investigation of biological multi-volume systems, characterized by different levels of complexity, ranging from cellular processes to population phenomena. BioSimWare can be run on singleprocessor personal computers (under Windows, Linux and Mac operating systems), as well as on parallel architectures exploiting MPI and CUDA libraries, or on distributed architectures such as grid. doi:10.1016/j.jbiotec.2010.09.831 [P-S.25] Towards a yeast cell cycle Sysbio model: Snf1/AMPK promotes S-phase entrance by controlling CLB5 transcription in budding yeast S. Pessina, S. Busnelli ∗ , M. Vanoni, L. Alberghina, P. Coccetti University of Milano-Bicocca, Italy Keywords: Snf1/AMPK; Cell cycle; Clb5; Saccharomyces cerevisiae The class of Snf1/AMPK (sucrose non-fermenting/AMPactivated protein kinase) is a central controller of cellular energy. This protein complex is conserved from lower to higher eukaryotes and it is commonly required for response to different cellular stresses. Saccharomyces cerevisiae Snf1 is a serine/threonine protein kinase that is able to reprogram transcription of metabolic genes required for growth upon glucose exhaustion. It also plays a role in chromatin remodelling and stress adaptation. Since it has been shown that in higher eukaryotes (mammalian cells and Drosophila melanogaster) the activation of AMPK causes a cell cycle arrest in G1 phase, we investigated the role of Snf1 kinase in the regulation of S-phase entrance in budding yeast. Contrary to the pattern observed in multicellular eukaryotes, we show that Snf1 is involved in the regulation of cell cycle progression by controlling the expression of the CLB5 mRNA, ensuring Clb5 protein accumulation and activity on Sld2 phosphorylation, a necessary requirement for the onset of DNA replication. In fact, in cells growing in 2% glu-
A system-level analysis of the action mechanism of small compounds displaying Ras inhibitory properties E. Sacco 1,∗ , A. D’Urzo 1 , A. Bargna 1 , V. Gaponenko 2 , F. Peri 1 , M. Vanoni 1 1
University of Milano-Bicocca, Dept. of Biotechnology and Bioscience, Italy 2 University of Illinois at Chicago, Dept of Biochemistry and Molecular Genetics, United States Keywords: Ras inhibitors; Ras/GEF cycle; Mathematical model Ras GTPases cycle between inactive GDP-bound state and active GTP-bound state to modulate a diverse array of processes involved in cellular growth control. Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. Recently water-soluble Ras inhibitors were synthesized and in vivo experiments show that the addition of compounds in the culture medium of normal and kras-transformed fibroblasts induces a dose-dependent decrease in proliferative potential. The compounds inhibit in a dose-dependent manner GEFcatalyzed dissociation of the guanine nucleotide from Ras and the entrance of the new nucleotide. The action mechanism of compounds is being analyzed in detail on both wild type and mutant Ras using both computational (docking and molecular modelling) and biochemical and biophysical data including calorimetric assays (ITC), SPR and mass spectrometry experiments. By using a computer model describing the possible interaction of the compounds with the Ras/GEF cycle (based on a model of the Ras/GEF cycle published by Lenzen et al., 1998) we are evaluating alternative models for the action of the compounds. The choice of unknown parameters for the simulations is constrained by above described experiments. This integrated computational and experimental system-level approach should prove valuable in fine characterization of drug mechanism and in the development of novel drugs with improved selectivity and /or efficacy. doi:10.1016/j.jbiotec.2010.09.833
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
cose synthetic media, deletion of the SNF1 gene yields a severe slow growth phenotype and a specific delay in the execution of the G1 to S transition, while the expression of the non-phosphorylatable mutant protein Snf1-T210A gave only a partial complementation of the growth rate. Thus we provided a previously unrecognized role for Snf1 in regulating the S-phase entrance, the area of major relevance for cell cycle control in budding yeast, and we showed that this role is partially independent from Snf1 activation. Our experimental results reported above are part of a modular systems biology approach, developped by our group, that relies on molecular analysis of perturbed cell cycle for regulatory interaction elucidation and networks identification in order to construct a comprehensive mathematical model of the yeast cell cycle. doi:10.1016/j.jbiotec.2010.09.832 Fig. 3.
[P-S.26] tems), DPP, tau-DPP, Stau-DPP. The software supports SBML format, and can automatically convert stochastic models into the corresponding deterministic formulation. Deterministic simulations can be performed by using different ODE solvers. Parameter estimation is executed through optimization methods as Particle Swarm Optimizer, Genetic Algorithms and Hill Climbing. Results: BioSimWare has been exploited for the investigation of many biological systems, such as the Ras/cAMP/PKA pathway in yeast, bacterial chemotaxis, a synthetic genetic oscillator coupled with quorum sensing, metapopulations, etc. (papers published in international journals). Discussion: The novelty of BioSimWare is that it allows the investigation of biological multi-volume systems, characterized by different levels of complexity, ranging from cellular processes to population phenomena. BioSimWare can be run on singleprocessor personal computers (under Windows, Linux and Mac operating systems), as well as on parallel architectures exploiting MPI and CUDA libraries, or on distributed architectures such as grid. doi:10.1016/j.jbiotec.2010.09.831 [P-S.25] Towards a yeast cell cycle Sysbio model: Snf1/AMPK promotes S-phase entrance by controlling CLB5 transcription in budding yeast S. Pessina, S. Busnelli ∗ , M. Vanoni, L. Alberghina, P. Coccetti University of Milano-Bicocca, Italy Keywords: Snf1/AMPK; Cell cycle; Clb5; Saccharomyces cerevisiae The class of Snf1/AMPK (sucrose non-fermenting/AMPactivated protein kinase) is a central controller of cellular energy. This protein complex is conserved from lower to higher eukaryotes and it is commonly required for response to different cellular stresses. Saccharomyces cerevisiae Snf1 is a serine/threonine protein kinase that is able to reprogram transcription of metabolic genes required for growth upon glucose exhaustion. It also plays a role in chromatin remodelling and stress adaptation. Since it has been shown that in higher eukaryotes (mammalian cells and Drosophila melanogaster) the activation of AMPK causes a cell cycle arrest in G1 phase, we investigated the role of Snf1 kinase in the regulation of S-phase entrance in budding yeast. Contrary to the pattern observed in multicellular eukaryotes, we show that Snf1 is involved in the regulation of cell cycle progression by controlling the expression of the CLB5 mRNA, ensuring Clb5 protein accumulation and activity on Sld2 phosphorylation, a necessary requirement for the onset of DNA replication. In fact, in cells growing in 2% glu-
A system-level analysis of the action mechanism of small compounds displaying Ras inhibitory properties E. Sacco 1,∗ , A. D’Urzo 1 , A. Bargna 1 , V. Gaponenko 2 , F. Peri 1 , M. Vanoni 1 1
University of Milano-Bicocca, Dept. of Biotechnology and Bioscience, Italy 2 University of Illinois at Chicago, Dept of Biochemistry and Molecular Genetics, United States Keywords: Ras inhibitors; Ras/GEF cycle; Mathematical model Ras GTPases cycle between inactive GDP-bound state and active GTP-bound state to modulate a diverse array of processes involved in cellular growth control. Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. Recently water-soluble Ras inhibitors were synthesized and in vivo experiments show that the addition of compounds in the culture medium of normal and kras-transformed fibroblasts induces a dose-dependent decrease in proliferative potential. The compounds inhibit in a dose-dependent manner GEFcatalyzed dissociation of the guanine nucleotide from Ras and the entrance of the new nucleotide. The action mechanism of compounds is being analyzed in detail on both wild type and mutant Ras using both computational (docking and molecular modelling) and biochemical and biophysical data including calorimetric assays (ITC), SPR and mass spectrometry experiments. By using a computer model describing the possible interaction of the compounds with the Ras/GEF cycle (based on a model of the Ras/GEF cycle published by Lenzen et al., 1998) we are evaluating alternative models for the action of the compounds. The choice of unknown parameters for the simulations is constrained by above described experiments. This integrated computational and experimental system-level approach should prove valuable in fine characterization of drug mechanism and in the development of novel drugs with improved selectivity and /or efficacy. doi:10.1016/j.jbiotec.2010.09.833