INFLAMMATION--IKAPPA B KINASE STRUCTURE FUNCTION STUDIES

最近已经确定了感染、慢性炎症和肿瘤发生之间的密切联系。由白细胞浸润引起的组织损伤是许多急慢性炎症性疾病的标志。NF-kappaB转录因子通过去除其细胞抑制剂（IkappaBs）而激活，并差异地激活促炎基因程序。IkappaB激酶（IKK）对IkappaB的磷酸化是这个过程中的关键调控事件。IKK是一种大分子量的蛋白质复合物，可被多种细胞外刺激激活。只有三个IKK亚基被明确识别，相关的激酶（IKKalpha和IKKbeta）和一个结构成分（IKKgamma）。实际上，我们对其结构和亚基化学计量学一无所知。我们假设存在一种未描述的IKK激酶（IKK- k），它特异性地将IKK与上游信号分子连接起来。我们体外激酶实验的初步结果表明，IKKgamma是IKK- k的靶标，IKKgamma的磷酸化导致其从IKK中部分去除，从而允许IKK。我们假设刺激诱导激活的IKK- k磷酸化IKKgamma的一个蛋白质，触发IKKgamma的构象变化，导致其从IKK复合体中部分去除，从而激活IKK，或者与IKK抑制分子有效相互作用，然后磷酸化IKKgamma，抑制剂离开复合体，从而激活IKK。为了验证这一假设，在Aim 1中，我们将使用免疫亲和纯化和磷酸肽定位/测序技术，从生物化学和生物物理上表征静息和刺激诱导细胞中IKK相关和释放IKKgamma的修饰。在目标2中，我们将使用传统的和免疫亲和的色谱法来纯化IKK-Ks，这些IKK-Ks在TNFalpha刺激后结合并磷酸化IKKgamma。为了确定细胞中是否存在不同类型的IKK复合物，在Aim 3中，我们将描述一些IKK亚基免疫亲和试剂，并开发功能性单克隆抗体（单链抗体（scFvs）），可用于识别和选择亚基特异性IKK复合物。这些也将在细胞内表达，以确定特定类型的IKK复合物在响应TNF或IL-1激活时的生理功能。揭示IKK复合体是如何响应环境信号而受到调节的，并确定其结构基础，将有助于未来开发专门针对IKK激活和NF-kappaB靶基因转录的药物。这些药物在治疗可导致癌症的急性和慢性炎症疾病方面具有很大的医学价值。

There has recently been identified a strong connection between infection, chronic inflammation and oncogenesis. Tissue damage resulting from leukocyte infiltration is the hallmark of many acute and chronic inflammatory diseases. NF-kappaB transcription factors are activated by removal of their cellular inhibitors, the IkappaBs, and differentially activate the proinflammatory gene program. IkappaB phosphorylation by the IkappaB kinase (IKK) is the key regulatory event in this process. IKK exists as a large molecular weight protein complex and is activated by a wide variety of extracellular stimuli. Only three IKK subunits have been identified unambiguously, the related kinases (IKKalpha and IKKbeta), and a structural component (IKKgamma). Virtually nothing is known about, structure or subunits stoichiometry. We hypothesize that an undescribed IKK kinase(s) (IKK-K) exists that specifically links the IKKs with upstream signaling molecules. Our Preliminary Results from in vitro kinase assays indicate IKKgamma is the target of the IKK-K and that phosphorylation of IKKgamma leads to its partial removal from IKK allowing of IKK. We hypothesize that stimulus-induced activated IKK-K phosphorylates a protein of the IKKgamma triggering a conformational change in IKKgamma causing either its partial removal from the IKK complex an activation of IKK or alternatively, an efficient interaction with an IKK inhibitory molecule and then phosphorylated IKKgamma and the inhibitor leave the complex resulting in activation of IKK. To test this hypothesis, in Aim 1 we will biochemically and biophysically characterize the modifications on IKK- associated and freed IKKgamma in resting and stimulus- induced cells using immunoaffinity purification and phospgopeptide mapping/sequencing techniques. In Aim 2, we will use both traditional and immuno-affinity, chromatography to purify IKK-Ks which associate and phpsphorylate IKKgamma following a TNFalpha stimulus. To determine if there are different types of IKK complexes in cells, in Aim 3 we will characterize a number of IKK subunits immunoaffinity reagents and also develop functional monoclonal antibodies (single chain antibodies (scFvs) that can be used to identify and select subunits-specific IKK complexes. These will also be expressed intracellularly to determine the physiological function of a particular type of IKK complex in response to TNF or IL-1 activation. Uncovering how the IKK complex is regulated in response to environmental cues and defining the structural basis for this will allow the future development of pharmaceuticals to specifically target IKK activation and NF-kappaB target gene transcription. These pharmaceuticals will be of great medical benefit in combating diseases of a acute and chronic inflammation, which can lead to cancer.