Mechanism of CD33 receptor signaling in Alzheimer disease

项目摘要 根据我长期的研究兴趣，了解细胞表面的跨膜(TM)信号 受体，目前的建议旨在阐明关键的免疫调节剂CD33的信号机制 小胶质细胞对淀粉样蛋白-β(A-β)多肽的清除。小胶质细胞的反应，驻留的吞噬细胞 中枢神经系统，to Aβ聚集体可以是吞噬和神经保护的，也可以是炎性的和 细胞毒性。这种影响使先天免疫反应成为阿尔茨海默病(AD)的主要决定因素 发病机制。小胶质细胞的激活状态和吞噬能力与信号转导有关。 CD33细胞表面受体。CD33失活促进吞噬并减轻β病理，识别 抑制CD33可作为治疗AD的一种有前景的途径。CD33属于唾液酸结合蛋白家族。 免疫球蛋白样受体(Siglecs)，由两个细胞外免疫球蛋白样结构域(IGV和 Ig C2)、单程跨膜(TM)结构域和胞质(CS)结构域。在这里，我们假设 IGV-IgC2-TM结构域-结构域取向和偶联在配体结合时发生变化，这传递了一种信号 (结构变化)到CS域。目标1为测试这一点奠定了结构和生化基础。 假肢。它建立表达系统来产生同位素标记的糖基化结构域，确定它们的 用核磁共振波谱分析了其结构、齐聚状态和膜浸泡情况。目标2跟踪结构 磷脂重组全长CD33中IGV与CS结构域的配基结合变化 来直接测试我们的信号假说。具体地说，配体引起的结构、结构域- 将使用核磁共振来确定结构域定向和耦合。CD33活动状态将在 将检测培养的巨噬细胞和基于结构的点突变，为药物提供靶点。 斑点CD33抑制。AIM 3检查CS域和下行信令之间的链接。这个 已建立的CD33结合蛋白的结合基序和亲和力将通过ITC和核磁共振确定。这个或者- 组织胞质配体的结合层次，鉴定其结合序列与Se- 阻断在受体激活时经历结构扰动的序列，并确定额外的CD33可以- 用于药物干预的双日期序列。而细胞表面受体片段与细胞外 配基结合结构域已经在原子分辨率下进行了研究，CD33相对较小的尺寸允许第一 对溶液中完整的这种受体的研究。描述了它在原子分辨率下的完整信号机制 为受体生物学建立了一个范例，并为Siglec生物学提供了新的见解。直接瞄准 β淀粉样蛋白作为阿尔茨海默病的治疗在很大程度上是不成功的，我们提供了细胞验证的CD33靶点 可转化为治疗学的生物学，以利用优化先天免疫的巨大潜力 对公元前战斗的回应。

Project Summary Pursuant to my long-term research interest, understanding the transmembrane (TM) signaling of cell surface receptors, the present proposal aims to elucidate the signaling mechanism of CD33, a key immune modulator of amyloid-β (Aβ) peptide clearance by microglia. The response of microglia, the resident phagocytes of the central nervous system, to Aβ aggregates can be either phagocytotic and neuroprotective or inflammatory and cytotoxic. This influence makes the innate immune response a major determinant of Alzheimer disease (AD) pathogenesis. The activation state and phagocytosis capability of microglia correlate with signaling by the CD33 cell-surface receptor. CD33 inactivation promotes phagocytosis and mitigates Aβ pathology, identifying CD33 inhibition as a promising therapeutic AD avenue. CD33 belongs to the family of sialic acid-binding im- munoglobulin-like receptor (Siglecs) and consists of two extracellular immunoglobulin-like domains (IgV and IgC2), a single-pass transmembrane (TM) domain and a cytosolic (CS) domain. Here, we hypothesize that the IgV-IgC2-TM domain-domain orientations and couplings change upon ligand binding, which transmits a signal (structural change) to the CS domains. Aim 1 lays the structural and biochemical groundwork to test this hy- pothesis. It establishes expression systems to produce isotope-labeled, glycosylated domains, determines their structure, oligomerization state and membrane immersion by NMR spectroscopy. Aim 2 traces the structural changes upon ligand binding from IgV to the CS domain in full-length CD33 reconstituted in phospholipid bicelles to directly test our signaling hypothesis. Specifically, ligand-induced changes in structure, domain- domain orientations and couplings will be established using NMR. The CD33 activity state will be verified in cultured macrophages and structure-based point mutations will be examined to provide target sites for phar- macological CD33 inhibition. Aim 3 examines the link between the CS domain and downstream signaling. The binding motif and affinity of established CD33-binding proteins will be determined by ITC and NMR. This or- ganizes the binding hierarchy of cytosolic ligands, identifies binders whose binding sequences overlap with se- quences that experience structural perturbations upon receptor activation, and identifies additional CD33 can- didate sequences for pharmacological intervention. While fragments of cell surface receptors with extracellular ligand-binding domains have been studied at atomic resolution, the relatively small size of CD33 allows the first study of an entire such receptor in solution. Delineating its complete signaling mechanism at atomic resolution establishes a paradigm for receptor biology and provides novel insight into Siglec biology. The direct targeting of Aβ amyloid as AD therapy has been largely unsuccessful and we provide CD33 target sites verified by cell biology that can be translated to therapeutics to harness the great potential of optimizing the innate immune response to combat AD.