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

核因子-kB家族转录因子控制细胞内和细胞内 信号、细胞应激反应、细胞生长、存活和凋亡。在静息细胞中，核因子-kB二聚体 通过与一个家族的相互作用将转录激活潜力隔离在细胞质中 Kappa B蛋白抑制物(LkB)。在大量不同刺激的作用下， 抑制物被磷酸化、泛化和降解，释放了核转录因子-kB核定位信号 以核因子-kB为靶点进入细胞核。靶基因的差异转录激活与 可用数学模型描述的对核因子-kB活性的时间控制。总体目标1，我们 将探索核因子-kB转录因子与其抑制物之间相互作用的动力学。 LkBalpha/NF-kB相互作用的结合动力学和热力学将与理论相关联 在相互作用中是否以及在哪里折叠与结合相耦合的预测。的新结构 家庭成员之间形成的复杂关系将得到解决，发生在 复杂的队形将被分析。实验将更好地将数学模型参数化。 在总体目标2中，我们将探索自由lkBalpha的结构和功能，并努力理解 它的部分折叠结构对它的任何功能是否重要。游离lkBalpha在体外具有边缘 热力学稳定性，可被泛素非依赖性蛋白酶体降解 降解途径。一组突变体将被分析热力学稳定性和体外和 体内蛋白酶体降解率。在总体目标3中，我们将开发新的综合方法 它跨越了从硅学理论到体外生化和生物物理实验的界限 核因子-kB信号网络的体内特性。理论算法将发展到更多 从部分折叠蛋白质的低分辨率实验数据准确预测结构 合唱团。我们独特的理论，体外生化和生物物理特征的结合， 体内研究将使我们能够通过系统地干扰核因子-kB来绘制地形图 信令网络。因此，NF-kB/lkBalpha信号系统代表了一个独特的例子，其中深度 对蛋白质相互作用动力学的生物物理理解可以定量地与 紧急生物反应。

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 (lkBs). Following the action of a large number of different stimuli, the inhibitor is phosphorylated, ubiquinated, and degraded, freeing the NF-kB 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 lkBalpha /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 lkBalpha and endeavor to understand whether its partially folded structure is important for any of its functions. Free lkBalpha 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/lkBalpha 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.