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

描述(由申请人提供)： 核因子-kB家族转录因子调控细胞内和细胞内信号传导、细胞应激反应、细胞生长、存活和凋亡。在静息细胞中，具有转录激活潜力的核因子-kB二聚体通过与一系列kappa B蛋白抑制物(LkB)相互作用而被隔离在细胞质中。在大量不同刺激物的作用下，抑制物被磷酸化、泛化和降解，将靶向核转录因子-kB的核定位信号释放到细胞核。靶基因的差异转录激活与核因子-kB活性的时间控制有关，这可以用数学模型来描述。在总体目标1中，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预测相关联，即在相互作用中折叠是否以及在哪里与结合相耦合。将解决家庭成员之间形成的复合体的新结构，并分析复合体形成时发生的动力学变化。实验将更好地将数学模型参数化。在整个目标2中，我们将探索自由lkBalpha的结构和功能，并努力了解其部分折叠结构是否对其任何功能重要。游离的lkBalpha在体外具有边际热力学稳定性，可能通过泛素非依赖的蛋白酶体降解途径在细胞内被降解。将对一组突变体进行热力学稳定性和体外和体内蛋白酶体降解率的分析。在总体目标3中，我们将开发新的综合方法，从电子理论到体外生化和生物物理实验，再到核因子-kB信号网络的体内特性。将开发理论算法，从部分折叠的蛋白质系综低分辨率实验数据中更准确地预测结构。我们独特的理论、体外生化和生物物理特征以及体内研究的结合，将使我们能够通过系统地干扰NF-kB信号网络来绘制图景。因此，NF-kB/lkBalpha信号系统代表了一个独特的例子，在这个例子中，对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。

DESCRIPTION (provided by applicant): 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.