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 kappa B (NF-kB) 转录因子家族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家族的相互作用而被隔离在细胞质中 kappa B 蛋白 (lkB) 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 它将 NF-kB 靶向细胞核。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-kB 转录因子与其抑制剂之间相互作用的动态。 lkBalpha /NF-kB 相互作用的结合动力学和热力学将与理论相关 预测折叠是否以及在何处与相互作用中的结合耦合。新的结构 家庭成员之间形成的情结将得到解决，家庭成员之间发生的动态变化将得到解决。 将分析复杂的形成。实验将更好地参数化数学模型。 在总体目标 2 中，我们将探索免费 lkBalpha 的结构和功能，并努力理解 其部分折叠结构对其任何功能是否重要。游离 lkBalpha 在体外具有边缘性 热力学稳定性，可被不依赖于泛素的蛋白酶体在细胞内降解 降解途径。将分析一组突变体的热力学稳定性以及体外和 体内蛋白酶体降解率。在总体目标 3 中，我们将开发新颖的综合方法 跨越了从计算机理论到体外生化和生物物理实验的界限 NF-kB 信号网络的体内特性。理论算法将发展到更多 根据部分折叠蛋白质的低分辨率实验数据准确预测结构 合奏团。我们独特地将理论、体外生化和生物物理表征相结合， 体内研究将使我们能够通过 NF-kB 的系统扰动来绘制景观图 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学理解可以定量地与 紧急生物反应。