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: 8459436
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 169.99万
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8459436
• 关键词: Address; Affect; Affinity; Animal Model; Animals; Ankyrin Repeat; Apoptosis; Behavior; Binding; Binding Sites; Biochemical; Biological; Biophysics; C-terminal; Cell physiology; Cells; Cellular Stress Response; Cellular biology; Complex; Computer Simulation; Coupled; Cytoplasm; DNA Binding; Data; Development; Disease; Dissociation; Dose; Ensure; Equilibrium; Family; Family member; Genes; Genetic Transcription; Growth; Half-Life; Homo; I-kappa B Proteins; Immune response; In Vitro; Interphase Cell; Kinetics; Link; Malignant Neoplasms; Maps; Measurement; Measures; Minor; Molecular; Molecular Chaperones; Motion; NF-kappa B; Pathway interactions; Phosphotransferases; Play; Principal Investigator; Process; Protein Isoforms; Protein Region; Proteins; Recruitment Activity; Regulation; Resolution; Response to stimulus physiology; Role; Signal Pathway; Signal Transduction; Site; Specificity; Stimulus; Structure; Suggestion; System; T-Lymphocyte; TNFRSF5 gene; Theoretical Studies; Thermodynamics; Time; Transcriptional Activation; Ubiquitin; Work; biological systems; cell growth; cytokine; dimer; in vivo; inhibitor/antagonist; insight; mathematical model; multicatalytic endopeptidase complex; mutant; preference; programs; protein folding; protein protein interaction; research study; response; single molecule; single-molecule FRET; theories; transcription factor

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.

描述（由申请人提供）：该程序项目采用了非常广泛的方法，以了解跨时间尺度的综合行为，从原子分辨率到核因子Kappa B（NF？b）转录因子信号系统的全部动物。 NF？BS控制细胞应激反应，细胞生长，存活和凋亡。系统控制是通过相互作用来实现的，即Kappa B蛋白（I？bs）的抑制剂家族，该家族在细胞质中隔离了NF？B家族成员，该家族有望快速激活。实验和数学建模表明，游离抑制剂的快速降解可实现低游离抑制剂浓度和鲁棒的信号反应。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。在总体目标1中，我们将探讨规范抑制剂的降解速率如何控制信号传导。通过停止流量和T型动力学折叠动力学，关于折叠途径，NMR动力学的理论研究以及“ DeGrons”的识别将共同解决这一目标。在总体目标2中，我们将探讨信号在动力学控制下的方式。我们发现i？b？促进NF？b从转录位点解离（“剥离”）。将使用缺乏“剥离”的突变体在细胞中分析这种现象，该机制将通过理论研究预测，动力学将通过单分子研究来衡量，NMR将研究三元复合物的结构，并将其作用纳入转录激活动态动力学动力学。在总体目标3中，我们将探讨I？bs稳定某些NF？b Homo和异二聚体，并影响刺激反应的特异性。某些复合物激活了特定的基因，但是分子机制，结合亲和力，抑制剂的“折叠”以及在细胞中的作用仍然不完整。我们的多尺度，定量组合理论，体外生化和生物物理特征以及体内研究将使我们能够通过系统地扰动蛋白质相互作用动态来绘制景观，并可以与新兴的生物学反应进行定量链接。