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

描述(由申请者提供)：该计划项目应用了非常广泛的方法来了解跨时间尺度和从原子分辨率到整个动物的核因子kappaB(NFκB)家族转录因子信号系统的整合行为。核因子κB调控细胞应激反应、细胞生长、存活和凋亡。系统控制是通过Kappa B蛋白抑制物家族(IκB)的相互作用来完成的，它们将NFκB家族成员隔离在细胞质中，准备快速激活。实验和数学模型表明，游离型缓蚀剂的快速降解实现了低游离型缓蚀剂浓度和稳健的信号响应。蛋白质区域的耦合折叠和结合似乎对确定降解速率和结合动力学至关重要。在总体目标1中，我们将探索典型抑制剂的降解率如何控制信号传递。停流和T-跳跃的折叠动力学、折叠路径的理论研究、核磁共振动力学以及“退化子”的识别将共同解决这一目标。在整个AIM 2中，我们将探索信号在运动控制下的方式。我们发现IκBα促进了NFκB从转录位点上的解离(“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来预测，动力学将通过单分子研究来测量，三元复合体的结构将通过核磁共振来研究，并将纳入随机性对转录激活动力学的影响。在整个目标3中，我们将探索IκB稳定某些影响刺激反应特异性的NFκB同源和异源二聚体的想法。某些复合体激活了特定的基因，但其分子机制、结合亲和力、抑制物的“折叠性”以及在细胞中的作用仍不完全。我们的多尺度、定量的理论、体外生化和生物物理表征以及体内研究将使我们能够通过系统扰动蛋白质相互作用动力学来绘制景观图，这些动力学可以定量地与紧急生物反应联系起来。

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.