Structure-Function Studies & Molecular Interactions of Chemokines

描述（由申请人提供）：趋化因子及其受体以其在免疫监视中的作用而闻名，它们控制白细胞的迁移和激活，以解决生理异常，如感染和癌症。然而，这些蛋白的不适当调控与包括炎症性疾病、动脉粥样硬化、癌症和艾滋病在内的大量病理有关。因此，了解它们如何发挥作用，以开发药物来阻断它们的活动，是非常有意义的。近年来，趋化因子诱导的细胞迁移是一个复杂的多步骤过程，涉及许多不同的相互作用，不仅是趋化因子与趋化因子受体的相互作用，还包括与糖胺聚糖（GAGs）的相互作用，以及通过同源和异齐聚化相互作用。gag是一种结构多样的线性碳水化合物，通常作为蛋白聚糖附着在膜结合蛋白锚点上。这些相互作用涉及局部隔离细胞表面的趋化因子，阻止扩散，因此趋化因子为迁移细胞提供方向线索。趋化因子的其他作用：GAG相互作用包括跨细胞运输，调节寡聚化与尚未明确表征的信号后果，以及调节受体结合和激活。由于GAG结构高度多样化，与趋化因子的相互作用可能显著促进细胞迁移的特异性和微调，远远超出趋化因子与受体的相互作用。然而，目前关于趋化因子GAG复合物的结构及其确切的功能后果的信息很少。该建议的一个主要假设是，通过趋化因子在GAGs上的寡聚化或gag诱导的非结构化结构域折叠产生的结构可塑性是功能特异性/多样性的机制，并且通过寡聚化或未折叠状态结构的变化，给定的趋化因子可能识别不同的GAGs。第二种假设是，一些趋化因子通过同源和异质寡聚化动态改变其结构，这可能控制细胞迁移的其他步骤，除了固定在gag上。目的1涉及对趋化因子：GAG复合物的结构研究：(i)鉴定优先识别特定趋化因子的GAG并确定特异性水平；（ii）表征结合亲和力和相互作用机制（例如，非结构化结构域的寡聚化或折叠）；（iii）确定从小的低聚物到大的低聚物在柔性gag上组装的配合物的跨尺度结构信息。为了补充这些研究，在Aim 2中，结构信息将与功能相关联，以探索趋化因子寡聚化和GAG相互作用的动态变化如何协调细胞迁移的离散顺序步骤：(i)胞吞作用和细胞在GAG上的表面呈现，（ii）白细胞阻滞，（iii） transmigration，以及（iv）细胞防御反应的激活。总之，这些研究将为通过GAG结合和寡聚化调节趋化因子功能提供新的见解，其中结构可塑性可能发挥重要作用。

DESCRIPTION (provided by applicant): Chemokines and their receptor are best known for their role in immune surveillance, where they control the migration and activation of leukocytes to resolve physiological abnormalities such as infection and cancer. However, inappropriate regulation of these proteins is associated with an extraordinary number of pathologies including inflammatory disease, atherosclerosis, cancer and AIDS. Thus there is significant interest in understanding how they function in order to develop drugs to block their activities. In recent years, it has become clear that chemokine-induced cell migration is a complex multistep process involving many different interactions, not only of chemokines with chemokine receptors, but also with glycosaminoglycans (GAGs) and with each other through homo- and hetero-oligomerization. GAGs are structurally diverse linear carbohydrates that are often attached to membrane bound protein anchors as proteoglycans. These interactions are involved in locally sequestering chemokines on cell surfaces, preventing diffusion so that chemokines provide directional cues for migrating cells. Other roles for chemokine:GAG interactions include transport across cells, regulation of oligomerization with as yet poorly characterized signaling consequences, and regulation of receptor binding and activation. Since GAG structures are highly diverse, interactions with chemokines may significantly contribute to the specificity and fine tuning of cell migration, well beyond the chemokine:receptor interaction. However, at present, there is little structural information about chemokine:GAG complexes and their precise functional consequences. An overarching hypothesis of this proposal, is that structural plasticity through oligomerization of chemokines on GAGs, or GAG-induced folding of unstructured domains, are mechanisms of functional specificity/diversity, and that through changes in oligomerization or unfolded state structure, a given chemokine may recognize different GAGs. A second hypothesis is that some chemokines dynamically change their structures through homo- and hetero-oligomerization which may control other steps in cell migration in addition to immobilization on GAGs. Aim 1 involves structural studies of chemokine:GAG complexes to (i) identify GAGs that preferentially recognize specific chemokines and determine the level of specificity; (ii) characterize binding affinities and interaction mechanisms (e.g. oligomerization or folding of unstructured domains); and (iii) determine trans-scale structural information of complexes from small oligomers through larger oligomeric assemblies on flexible GAGs. To complement these studies, in Aim 2, structural information will be correlated with function to probe how dynamic changes in chemokine oligomerization and GAG interactions coordinate discrete sequential steps of cell migration: (i) transcytosis and cell surface presentation on GAGs, (ii) leukocyte arrest (iii) transmigration, and (iv) activation of cellular defense responses. Together these studies will provide novel insight into the regulation of chemokine function through GAG- binding and oligomerization, where structural plasticity is likely to play a significant role.