Processing of NF-kB2/P100

描述(申请人提供)：核因子-B(核因子-B)二聚体转录因子家族通过调节直接参与这些生物过程的广泛基因的表达来控制关键的生物事件，如先天和免疫反应，以及细胞增殖和程序性细胞死亡。核因子-βB家族由p50(核因子-kappaB1)、p52(核因子-kappaB2)、p65(RelA)、c-rel和RelB五个亚基组成，它们以组合的方式形成功能性的核因子-kappaB二聚体。P52/RelB异源二聚体是一种组合二聚体，是淋巴细胞中的关键转录因子。P52是由前体蛋白P100通过蛋白酶体对特定刺激的反应而产生的。蛋白酶体对分子的这种不寻常的、依赖于信号的部分降解的机制尚不清楚。 本申请的重点是研究p100到p52的信号相关处理是如何发生的。我们提出了一个模型来解释这一处理事件背后的机制。众所周知，RelB与其自身或与p65和c-Rel无关。然而，它优先与P100相关联。这些观察结果使我们假设，与RelB的异二聚化是P100被加工成P52和形成P52/RelB杂二聚体的先决条件。这项提议将测试与RelB的结合是否是P100加工的必需步骤，以及与其他NF-kappaB二聚体相比，P100/RelB二聚体是否在结构上是独特的二聚体。我们的初步实验表明，在RelB缺陷细胞中，P100对P52的处理被阻断。我们进一步揭示了RelB的二聚化结构域是相对不稳定的，这种缺乏稳定性可能是形成非活性P100/RelB异质二聚体的关键。这一建议将结合体外生化和X射线结晶学研究与体内实验，使用各种核因子-kappaB亚基敲除细胞，以了解一种重要的核因子-kappaB异源二聚体的激活机制。

DESCRIPTION (provided by applicant): The nuclear factor-?B (NF-?B) family of dimeric transcription factors controls key biological events such as innate and immune responses, and cell proliferation and programmed cell death, by regulating the expression of a wide range of genes that are directly involved in those biological processes. The NF-?B family is comprised of five subunits, p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA), c-Rel and RelB, which form functional NF-kappaB dimers in a combinatorial manner. The p52/RelB heterodimer is one of the combinatorial dimers which is a key player transcription factor in lymphoid cells. p52 is derived from the precursor protein p100 through processing by the proteasome in response to specific stimuli. The mechanism of this unusual, signal-dependent partial degradation of a molecule by the proteasome is unknown. The focus of this application is to study how the signal-dependent processing of p 100 to p52 occurs. We propose a model to explain the mechanism that underlies this processing event. It is known that RelB does not associate with itself or with p65 and c-Rel. However, it preferentially associates with p100. These observations lead us to hypothesize that hetero-dimerization with RelB is a pre-requisite event for p100 to be processed into p52 and the formation of the p52/RelB heterodimer. This proposal will test whether or not association with RelB is an obligatory step for p100 processing, and whether the p100/RelB dimer is a structurally unique dimer compared to other NF-kappaB dimers. Our preliminary experiments have shown that in RelB deficient cells, p100 processing to p52 is blocked. We have further revealed that the dimerization domain of RelB is relatively unstable and this lack of stability might be critical for the formation of the inactive p100/RelB heterodimer. This proposal will integrate in vitro biochemical and X-ray crystallographic studies with in vivo experiments using various NF-kappaB subunit knock out cells to understand the mechanism of activation of an important NF-kappaB heterodimer.