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

描述（由申请人提供）：本计划项目应用了非常广泛的方法来理解转录因子信号系统的核因子κ B（NFκB）家族在整个动物中跨时间尺度和从原子分辨率的综合行为。NFκ B控制细胞应激反应、细胞生长、存活和细胞凋亡。系统控制是通过与kappa B蛋白（Iκ B）抑制剂家族相互作用来实现的，该抑制剂家族将NFκB家族成员隔离在细胞质中，准备快速激活。实验和数学建模表明，游离抑制剂的快速降解实现了低游离抑制剂浓度和稳健的信号响应。蛋白质区域的耦合折叠和结合对于定义降解速率和结合动力学似乎至关重要。在总体AIM 1中，我们将探索典型抑制剂的降解速率如何控制信号传导。通过停止流动和T-跳跃的折叠动力学、折叠途径的理论研究、NMR动力学和“降解决定子”的鉴定将共同解决这一目标。在总体目标2中，我们将探索信号传导受动力学控制的方式。我们发现IκBα促进NFκB从转录位点解离（“剥离”）。这种现象将在细胞中使用缺乏“剥离”的突变体进行分析，其机制将通过理论研究进行预测，动力学将通过单分子研究进行测量，三元复合物的结构将通过NMR进行研究，并且将纳入随机性对转录激活动力学的影响。在总体目标3中，我们将探讨Iκ B稳定某些NFκB同源和异源二聚体影响刺激反应特异性的观点。某些复合物激活特定的基因，但分子机制，结合亲和力，抑制剂的“折叠性”和在细胞中的作用仍然不完整。我们的多尺度，定量结合的理论，在体外生物化学和生物物理特性，并在体内的研究将使我们能够映射的景观系统扰动的蛋白质相互作用的动力学可以定量地联系到紧急的生物反应。

DESCRIPTION (provided by applicant): This Program Project applies an extraordinarily broad array of approaches to understand the integrated behavior across time scales and from atomic resolution to whole animals of the nuclear factor kappa B (NFκB) family of transcription factor signaling system. NFκBs control cellular stress responses, cell growth, survival, and apoptosis. System control is accomplished by interaction a family of inhibitors of kappa B proteins (IκBs) that sequester NFκB family members in the cytoplasm poised for rapid activation. Experiments and mathematical modeling showed that rapid degradation of free inhibitors achieves low free inhibitor concentrations and robust signal response. Coupled folding and binding of regions of the proteins appears critical for defining degradation rates and binding kinetics. In Overall AIM 1, we will explore how the degradation rate of the canonical inhibitors controls signaling. Folding kinetics by stopped flow and T-jump, theoretical studies on the folding pathways, NMR dynamics, and identification of the "degrons" will together address this aim. In Overall AIM 2, we will explore ways in which the signaling is under kinetic control. We have discovered that IκBα facilitates dissociation of NFκB from transcription sites ("stripping"). This phenomenon will be analyzed in cells using mutants deficient in "stripping", the mechanism will be predicted by theoretical studies, the kinetics will be measured by single molecule studies, the structures of ternary complexes will be studied by NMR and the effects of stochasticity on the kinetics of transcription activation will be incorporated. In Overall AIM 3, we will explore the idea that IκBs stabilize certain NFκB homo and heterodimers affecting the specificity of stimulus response. Certain complexes activate specific genes, yet the molecular mechanism, binding affinities, "foldedness" of the inhibitors, and roles in cells are still incomplete. Our multiscale, quantitative 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 protein interaction dynamics can be quantitatively linked to the emergent biological response.