INFLAMMATION--IKAPPA B KINASE STRUCTURE FUNCTION STUDIES

最近发现感染、慢性炎症和肿瘤发生之间有很强的联系。白细胞渗入引起的组织损伤是许多急慢性炎症性疾病的特征。核因子-kappaB转录因子通过去除其细胞抑制物IkappaB而被激活，并以不同的方式激活促炎基因程序。IkappaB激酶(IKK)对IkappaB的磷酸化是这一过程中的关键调控事件。IKK是一种大分子蛋白质复合体，可被多种细胞外刺激激活。只有三个IKK亚基被明确确定，即相关的激酶(IKKalpha和IKKbeta)和一个结构成分(IKKGamma)。事实上，人们对化学计量学的结构或亚基一无所知。我们假设存在一个未被描述的IKK激酶(S)(IKK-K)，它特异性地将IKK与上游信号分子联系起来。我们的体外激酶分析的初步结果表明，IKKGamma是IKK-K的靶标，并且IKKGamma的磷酸化导致其部分从IKK中去除，从而允许IKK。我们假设，刺激诱导激活的IKK-K磷酸化IKK伽马蛋白，触发IKK伽马的构象变化，导致其部分从IKK复合体中移除，激活IKK，或者，与IKK抑制分子有效相互作用，然后磷酸化的IKK伽马和抑制剂离开复合体，导致IKK激活。为了验证这一假设，在目标1中，我们将使用免疫亲和纯化和磷肽图/测序技术，在静息和刺激诱导的细胞中对IKK相关的和释放的IKKGamma的修饰进行生化和生物物理表征。在目标2中，我们将同时使用传统的和免疫亲和层析来纯化IKK-Ks，这些Ks在TNFpha刺激后结合和磷酸化IKKGamma。为了确定细胞中是否存在不同类型的IKK复合体，在目标3中，我们将表征一些IKK亚基免疫亲和试剂，并开发可用于识别和选择亚基特异性IKK复合体的功能性单抗(单链抗体)。这些也将在细胞内表达，以确定特定类型的IKK复合体在响应肿瘤坏死因子或白介素1激活时的生理功能。揭示IKK复合体是如何对环境信号做出反应并确定其结构基础的，将使未来的药物开发能够专门针对IKK激活和NF-kappaB靶基因转录。这些药物在对抗可能导致癌症的急慢性炎症疾病方面将有很大的医疗益处。

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.