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

描述（由申请人提供）：该计划项目适用于非常广泛的方法，以了解跨时间尺度的综合行为，从原子分辨率到整个动物的核因子κ B（NF？B）转录因子信号系统家族。NF？Bs控制细胞应激反应、细胞生长、存活和凋亡。系统控制是通过与κ B蛋白抑制剂家族（I？B）隔离NF？细胞质中的B家族成员准备快速激活。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。我们发现我？B？促进NF的解离？B从转录位点（“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，我们将探讨我？Bs稳定某些NF？影响刺激反应特异性的B同源和异源二聚体。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。 公共卫生相关性：转录因子的核因子κ B家族控制无数的细胞功能，包括生长调节，从而控制癌症、免疫应答和发育。数百种不同的基因是如何被开启和关闭的，目前还不清楚。我们的理论生物物理学，实验方法和细胞生物学的结合将提供一个深刻的理解这一重要系统。

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 (NF?B) family of transcription factor signaling system. NF?Bs control cellular stress responses, cell growth, survival, and apoptosis. System control is accomplished by interaction a family of inhibitors of kappa B proteins (I?Bs) that sequester NF?B 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 I?B? facilitates dissociation of NF?B 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 I?Bs stabilize certain NF?B 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. PUBLIC HEALTH RELEVANCE: The nuclear factor kappa B family of transcription factors controls a myriad of cellular functions including growth regulation and thus cancer, the immune response, and development. How the hundreds of different genes are turned on and off specifically is not yet understood. Our combination of theoretical biophysics, experimental approaches and cell biology will provide a deep understanding this important system.