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

IkB/NF-kB 计算机、体外和体内识别

• 批准号: 7924964
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 12.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924964
• 关键词: IkB; NF; kB; Recognition; Silico

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.

核因子κ B（NF-kB）家族的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，NF-kB二聚体 具有转录激活潜力的基因通过与一个家族的相互作用而被隔离在细胞质中。 κ B蛋白（lkBs）的抑制剂。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导向细胞核靶基因的差异转录激活与 NF-kB活性的时间控制可以通过数学模型来描述。在总体目标1中，我们 将探索NF-κ B转录因子及其抑制剂之间相互作用的动力学。 lkB α/NF-κ B相互作用的结合动力学和热力学将与理论结合动力学和热力学相关。 预测在相互作用中折叠是否以及在何处与结合相耦合。新结构 家庭成员之间形成的复合体将得到解决， 将分析复合物的形成。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lkB α的结构和功能，并奋进了解 其部分折叠的结构对于其任何功能是否重要。游离lkB α在体外具有边际 热力学稳定性，并可在细胞内降解的泛素非依赖性蛋白酶体 降解途径将分析一组突变体的热力学稳定性和体外稳定性， 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， NF-kB信号网络的体内性质。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 合奏。我们独特的理论结合，体外生物化学和生物物理表征， 体内研究将使我们能够通过系统地干扰NF-κ B来绘制景观 信令网因此，NF-kB/lkB α信号传导系统代表了一个独特的例子， 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