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: 7221873
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 113.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-07 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7221873
• 关键词: IkB; NF; kB; Recognition; Silico

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.

描述（由申请人提供）： 核因子κ B（NF-kB）家族的转录因子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（lkB）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-kB靶向细胞核的NF-kB核定位信号。靶基因的差异转录激活与NF-kB活性的时间控制相关，其可以通过数学模型来描述。在总体AIM 1中，我们将探索NF-kB转录因子及其抑制剂之间相互作用的动力学。结合动力学和热力学的lkB α/NF-κ B相互作用将相关的理论预测折叠是否和在哪里耦合到结合的相互作用。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lkBalpha的结构和功能，并奋进了解其部分折叠结构是否对其任何功能都很重要。游离的IkB α在体外具有边际热力学稳定性，并且可以通过不依赖于泛素的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，以更准确地预测结构的低分辨率实验数据的部分折叠蛋白质合奏。我们独特的理论，体外生物化学和生物物理特性，以及体内研究的结合，将使我们能够通过系统扰动NF-κ B信号网络来绘制景观。因此，NF-κ B/lkB α信号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学理解可以定量地与紧急的生物反应。