IkB/NF-kB Recognition In Silico, In Vitro and In Vivo

核因子κ B （NF-KB）家族转录因子控制细胞间和细胞内信号传导、细胞应激反应、细胞生长、存活和凋亡。在静息细胞中，具有转录激活电位的NF-KB二聚体通过与kappa B蛋白（mb）抑制剂家族的相互作用被隔离在细胞质中。在大量不同刺激的作用下，抑制剂被磷酸化、泛素化和降解，释放NF-KB核定位信号，将NF-KB靶向到细胞核。靶基因的差异转录激活与NF-KB活性的时间控制有关，这可以用数学模型来描述。在总体AIM 1中，我们将探讨

The nuclear factor kappa B (NF-KB) family of transcription factors controls inter- and intracellular signaling, cellular stress responses, cell growth, survival, and apoptosis. In resting cells, NF-KB dimers with transcription activation potential are sequestered in the cytoplasm by interaction with a family of inhibitors of kappa B proteins (MBs). Following the action of a large number of different stimuli, the inhibitor is phosphorylated, ubiquinated, and degraded, freeing the NF-icB nuclear localization signal which targets the NF-KB to the nucleus. Differential transcription activation of target genes is linked to temporal control of NF-KB activity that can be described by a mathematical model. In Overall AIM 1, we will explore the dynamics of the interaction between the NF-KB transcription factors and their inhibitors. Binding kinetics and thermodynamics for the iKBa/NF-KB interaction will be correlated to theoretical predictions of whether and where folding is coupled to binding in the interaction. New structures of the complexes formed between family members will be solved and dynamics changes that occur upon complex formation will be analyzed. The experiments will better parameterize the mathematical model. In Overall AIM 2, we will explore the structure and function of free MBa and endeavor to understand whether its partially folded structure is important for any of its functions. Free iKBa has marginal in vitro thermodynamic stability and may be degraded intracellularly by a ubiquitin-independent proteasome degradation pathway. A panel of mutants will be analyzed for thermodynamic stability and in vitro and in vivo proteasome degradation rates. In Overall AIM 3, we will develop novel integrative approaches that cross the boundaries from in silico theory to in vitro biochemical and biophysical experiments to the in vivo properties of the NF-KB signaling network. Theoretical algorithms will be developed to more accurately predict structures from low resolution experimental data on partially folded protein ensembles. Our unique combination of theory, in vitro biochemical and biophysical characterization, and in vivo studies will enable us to map the landscape by systematic perturbation of the NF-KB signaling network. Thus, the NF-KB/IicB signaling system represents a unique example where a deep biophysical understanding of the protein interaction dynamics can be quantitatively linked to the emergent biological response.