Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

涉及核因子 KAPPA B 的信号通路的调控

• 批准号: 9560551
• 负责人: michael j lenardo
• 金额: $ 30.31万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9560551
• 关键词: Adjustment Disorders; Affect; Antigen Receptors; B-Lymphocytes; Binding; Biochemical; Biochemical Genetics; Biological Products; Biology; Cell Proliferation; Cells; Chromatin; Complex; Cytoplasm; DNA; DNA Sequence; DNA-Binding Proteins; Data; Dimerization; Disease; Gene Components; Gene Expression; Gene Family; Gene Mutation; Genes; Genetic Transcription; HIV; Half-Life; Homo; Human; Immune; Immune Response Genes; Immune System Diseases; Immune response; Immune system; Immunologic Deficiency Syndromes; Impairment; Individual; Infectious Agent; Inflammatory; Kinetics; Lead; Lymphocyte; Malignant Neoplasms; Mass Spectrum Analysis; Medicine; Mutation; NF-kappa B; Nuclear; Output; Pathway interactions; Pharmacologic Substance; Phosphorylation; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Property; Protein Family; Protein Kinase; Protein Subunits; Proteins; REL Protein; Receptors, Antigen, B-Cell; Regulation; Regulator Genes; Role; Signal Pathway; Signal Transduction; Stimulus; System; T-Cell Receptor; T-Lymphocyte; TNFRSF5 gene; Temperature; Time; Ubiquitination; Work; biophysical techniques; combat; dimer; gene induction; genetic approach; genetic regulatory protein; human disease; insight; interest; light scattering; metaplastic cell transformation; novel strategies; p65; preference; programs; protein activation; protein complex; prototype; response; synaptotagmin I; transcription factor; v-rel Oncogenes

Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a p50 DNA binding protein and a p65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. Overall there are 5 Rel protein subunits, including p50 and p65, that form a variety of hetero- or homodimers that have been widely studied and have important roles in the immune system. Nevertheless, very little is known about the kinetics of NF-kB dimer formation or the stability or interconversion of dimerised Rel subunits. We therefore carried out a quantitative analysis of association and disassociation properties of purified p50 and p65 Rel subunits using light scattering, mass spectrometry, and other biophysical techniques. Our data show that at physiological temperatures, p50 or p65 homodimers efficiently exchange subunits with a half-life of less than 10 minutes. Even more interesting, we found a marked preference for the formation of the p50/p65 heterodimer, which kinetic analyses show was at least 10 times more stable than either homodimer. The implications of these findings are that specific DNA targets of either the p50 or p65 homodimers may be restricted to situations in which these subunits are expressed exclusively, or require additional chromatin factors or post-translational modifications. Together, our work indicates that cells can modulate NF-kB activity by finely modulating the relative proportions of the p50 and p65 proteins, which will determine the quantities of different homo- or hetero-dimers. This work thus provides a new quantitative understanding of Rel dimer distribution in the cell, which has important implications for our understanding of how this transcription factor system controls genes in the healthy and diseased immune system.

许多免疫反应基因的调节取决于称为Kappab的10 bp DNA序列。该序列由与REL癌基因有关的蛋白质因子系列结合。 原型转录复合物与序列的结合称为NF-kappab，通常被定义为P50 DNA结合蛋白与p65（rela）激活蛋白之间通常被称为I-kappab蛋白质的细胞质中隔离的p65（rela）激活蛋白。遵循对细胞的某些类型的刺激，一种称为I-kappab激酶的特定蛋白激酶复合物会导致I-kappab的磷酸化，然后其泛素化和降解。在可以释放NF-kappab的刺激中，在免疫反应过程中，抗原触发了T细胞受体（TCR）或B细胞受体（BCR）。然而，该转录因子在响应数百种不同诱导剂的诱导中诱导各种基因集中起作用。 总体而言，有5个REL蛋白亚基，包括P50和P65，它们形成了多种杂种或同型二聚体，这些异二聚体已被广泛研究并在免疫系统中具有重要作用。然而，关于NF-KB二聚体形成的动力学或二聚体二聚体的稳定性或相互转换的动力学知之甚少。因此，我们使用光散射，质谱和其他生物物理技术对纯化的P50和P65 Rel亚基的关联和分离特性进行了定量分析。我们的数据表明，在生理温度下，P50或P65同型二聚体有效地交换了不到10分钟的半衰期。更有趣的是，我们发现对P50/p65异二聚体的形成明显偏爱，动力学分析显示，这比任何一个同二聚体都比稳定性高10倍。这些发现的含义是，P50或P65同型二聚体的特定DNA靶标可能仅限于仅表达这些亚基的情况，或者需要其他染色质因子或翻译后修饰。 总之，我们的工作表明细胞可以通过细微调节P50和p65蛋白的相对比例来调节NF-KB活性，这将确定不同的同型或异方差二聚体的数量。 因此，这项工作为细胞中的Rel Dimer分布提供了新的定量理解，这对我们对该转录因子系统如何控制健康和患病免疫系统中基因的理解具有重要意义。