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

描述（由申请人提供）：本项目应用了一系列非常广泛的方法来理解核因子kappa B (NF？B)转录因子信号系统家族。NF吗?b控制细胞应激反应、细胞生长、存活和凋亡。系统控制是通过kappa B蛋白抑制剂家族(I？b)封存NF？B家族成员在细胞质中处于快速激活状态。实验和数学模型表明，快速降解游离抑制剂可获得较低的游离抑制剂浓度和较强的信号响应。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至关重要。在总体AIM 1中，我们将探讨典型抑制剂的降解率如何控制信号传导。通过停止流动和t跳的折叠动力学，折叠途径的理论研究，核磁共振动力学和“degrons”的识别将共同解决这一目标。在总体AIM 2中，我们将探索信号在动力学控制下的方式。我们发现I?B？促进NF?的解离B从转录位点（“剥离”）。这一现象将在缺乏“剥离”的突变体细胞中进行分析，其机制将通过理论研究预测，动力学将通过单分子研究测量，三元配合物的结构将通过核磁共振研究，并纳入随机性对转录激活动力学的影响。在总体AIM 3中，我们将探讨I？b稳定某些NF？B同性和异源二聚体影响刺激反应的特异性。某些复合物可以激活特定的基因，但抑制剂的分子机制、结合亲和力、“折叠性”以及在细胞中的作用仍然不完整。我们的多尺度，定量结合理论，体外生化和生物物理表征，以及体内研究将使我们能够绘制景观，通过系统扰动的蛋白质相互作用动态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 (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.