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

转录因子的核因子κ B（NF-κ B）家族控制胞内和胞间信号传导、细胞应激反应、细胞生长、存活和凋亡。在静息细胞中，具有转录激活潜力的NF-κ B二聚体通过与κ B蛋白（MB）抑制剂家族相互作用而被隔离在细胞质中。在大量不同刺激的作用下，抑制剂被磷酸化、泛素化和降解，释放出将NF-κ B靶向细胞核的NF-κ B核定位信号。靶基因的差异转录激活与NF-κ B活性的时间控制相关，其可以通过数学模型来描述。在总体目标1中，我们将探讨 NF-κ B转录因子及其抑制剂之间相互作用的动力学。iKBa/NF-κ B相互作用的结合动力学和热力学将与相互作用中折叠是否与结合偶联以及在何处与结合偶联的理论预测相关。将解决家庭成员之间形成的复合物的新结构，并分析复合物形成时发生的动力学变化。 实验结果将更好地对数学模型进行参数化。 在总体目标2中，我们将探索游离MBa的结构和功能，并奋进了解其部分折叠的结构是否对其功能很重要。游离iKBa在体外具有临界热力学稳定性，并可通过泛素非依赖性蛋白酶体降解途径在细胞内降解。将分析一组突变体的热力学稳定性以及体外和体内蛋白酶体降解速率。 在总体目标3中，我们将开发新的综合方法，从计算机理论到体外生物化学和生物物理实验，再到NF-κ B信号网络的体内特性。 将开发理论算法，以便从低分辨率更准确地预测结构 部分折叠的蛋白质集合体的实验数据。我们独特的理论结合，在体外 生物化学和生物物理表征以及体内研究将使我们能够通过NF-κ B信号传导网络的系统扰动来绘制景观。因此，NF-κ B/IicB信号传导系统代表了一个独特的例子，其中蛋白质相互作用动力学的深入生物物理理解可以定量地与紧急生物反应联系起来。

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.