Potential energy landscape and robustness of a gene regulatory network: Toggle switch

Finding a multidimensional potential landscape is the key for addressing important global issues, such as the robustness of cellular networks. We have uncovered the underlying potential energy landscape of a simple gene regulatory network: a toggle switch. This was realized by explicitly constructing the steady state probability of the gene switch in the protein concentration space in the presence of the intrinsic statistical fluctuations due to the small number of proteins in the cell. We explored the global phase space for the system. We found that the protein synthesis rate and the unbinding rate of proteins to the gene were small relative to the protein degradation rate; the gene switch is monostable with only one stable basin of attraction. When both the protein synthesis rate and the unbinding rate of proteins to the gene are large compared with the protein degradation rate, two global basins of attraction emerge for a toggle switch. These basins correspond to the biologically stable functional states. The potential energy barrier between the two basins determines the time scale of conversion from one to the other. We found as the protein synthesis rate and protein unbinding rate to the gene relative to the protein degradation rate became larger, the potential energy barrier became larger. This also corresponded to systems with less noise or the fluctuations on the protein numbers.

Dynamics and intrinsic statistical fluctuations of a gene switch

We studied a simple gene regulatory network, the toggle switch. Specifically, we examined the means and statistical fluctuations in numbers of proteins. We found that when omega, the ratio of rates of protein-gene unbinding to protein degradation, was between similar to 10(-3) and similar to 10, the fluctuations were much larger than those we would have expected from Poisson statistics. In addition, we examined characteristic time values for system relaxation and found both that they increased with omega and that they have significant phase transition effects, with a secondary time scale appearing near the boundary between bistable and other phases. Last, we discuss the bistability of the toggle switch.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the alpha and beta domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1 alpha (HIF1 alpha), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1 alpha and angiogenesis, possibly via direct binding and stabilization of pVHL. [Cancer Res 2009;69(6):2599-606]

High resolution mass spectrometric alveolar proteomics: Identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micro preparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products.