Biophysical and biochemical techniques for the analysis and targeting of the Fc receptor supramolecular complex

我们将分析一个模型受体，Fc受体，通过质谱和活细胞显微镜在IgG-微珠结合，识别和激活的时间过程。受体是细胞表面表达的蛋白质，控制对体内循环的信使的反应，是治疗疾病和疼痛的关键靶点。我们非常需要了解和比较细胞表面的受体，并创造针对它们的新的和特异性的药物。对组织损伤（如伤口和感染）的许多反应涉及称为巨噬细胞的特殊细胞的激活，这些细胞反过来触发炎症反应。在大多数情况下，炎症是有益的，但偶尔异常的炎症反应可以引发疾病过程，例如，关节炎性关节炎、动脉粥样硬化性炎症，或类似癌症和阿尔茨海默氏痴呆症。标记入侵细菌以进行破坏的人免疫球蛋白G（IgG）或其可结晶片段（Fc）可用于从巨噬细胞系（包括人分化的U937细胞和RAW巨噬细胞）捕获它们的受体和/或共受体。我们的目标是激活并随后捕获非炎性的单价Fc γ受体-IgG-S-S-生物素复合物，用于在链霉亲和素上收集，并通过一步简单还原释放。聚集的IgG-微珠将直接从活细胞表面活化并捕获活化的吞噬复合物，用于通过质谱法分离和表征。用培养的巨噬细胞对配体包被的微珠进行质谱分析、生物化学测定和活细胞激光显微镜检查，将揭示并确认调节吞噬作用和自由基产生的Fc活化机制。我们观察到Fc γ、α、μ受体与Fc样受体（Fc γ）、白细胞免疫球蛋白样受体（LILR）、杀伤细胞（KIR）和杀伤凝集素受体（KLR）的特异性同种型紧密相关，这些受体一起存在于IgG-微珠上的超分子复合物中，而不是oxLDL或抗CD 36微珠上。沉默针对Fc受体和共受体的特异性发现的复合物的RNA将证明共受体在吞噬作用或自由基产生中的作用。巨噬细胞和嗜中性粒细胞是吞噬细胞，因此具有释放自由基如超氧化物（O2-*）的能力，其在吞噬和破坏IgG-细菌复合物期间引起H2 O2的形成。然而，迄今为止，几乎没有绝对定量的方法可用于测量大多数细胞类型的少量H2 O2产物。我们已经表明，可以在细胞生长培养基中创建标准曲线和H2 O2测定，并且将检测H2 O2，使细胞响应IgG和其他配体、药物或受体siRNA而降低至约1皮科摩尔水平。Fc受体CD 64将在幼稚细胞的表面上表达，幼稚细胞将被固定并用作色谱柱以捕获受体配体IgG或Fc肽（相对于对照幼稚细胞）。在IgG-Fc受体复合物中发现了许多表皮生长因子（EGF）、白细胞介素（IL）和集落刺激因子（CSF）以及FLT 3R的受体。发现FLT 3配体调节IgG包被的微珠的吞噬作用，我们将测试其对IgG包被的微珠的吞噬作用和过氧化氢产生的影响。我们将提供一种新的和强大的方法来捕获细胞表面受体和辅助受体，测量它们在自由基释放和吞噬中的作用，然后快速筛选和测试它们的蛋白质配体，可能作为针对受体特异性炎症功能的生物药物。

We will analyze a model receptor, the Fc receptor, by mass spectrometry and live-cell microscopy over the time course of IgG-microbead binding, recognition and activation. Receptors are proteins expressed on the surface of cells that control the responses to messengers circulating in the body and are critical targets in the treatment of disease and pain. There is a great need to understand and compare receptors on the surface of cells and to create new and specific drugs against them. Many responses to tissue injury such as wounds and infection involve the activation of special cells called macrophages that in turn trigger an inflammatory response. In most instances, inflammation is beneficial, but occasionally an aberrant inflammatory response can initiate a disease process, e.g., arthritic inflammation of the joints, atherosclerotic inflammation of the arteries, or like cancer and Alzheimer's dementia. Human immunoglobulin G (IgG) or its fragment crystalizable (Fc) that flags invading bacteria for destruction may be used to capture their receptors and/or co-receptors from macrophage cell lines including human differentiated U937 cells and RAW macrophages. We aim to activate and then capture the non-inflammatory, monovalent Fc gamma receptor-IgG-S-S-biotin complex for collection over strepavidin, and release by a simple reduction in one step. Aggregated IgG-microbeads will activate and capture the activated phagocytic complex directly from the surface of living cells for isolation and characterization by mass spectrometry. Mass spectrometry, biochemical assays and live-cell laser microscopy of the ligand coated microbeads with cultured macrophages will reveal and confirm the mechanisms of Fc activation that regulate phagocytosis and free radical production. We observed the Fc gamma, epsilon, alpha, mu receptors were tightly associated with specific isoforms of Fc-like receptors (FCLR), Leukocyte Immunoglobulin-Like receptors (LILR), killer cell (KIR) and Killer lectin receptors (KLR) that were present together in a supramolecular complex on IgG-, but not oxLDL or anti CD36, microbeads. Silencing RNA against the specifically discovered complex of Fc receptors and co-receptors will demonstrate the role of the co-receptors in phagocytosis or free radical production. Macrophages and neutrophils are phagocytes and thus possess the capacity to release free radicals like superoxide (O2-*) that causes the formation of H2O2 during the engulfment and destruction of IgG-bacteria complexes. However, there are few absolute quantitative methods available for measuring the low amounts of H2O2 products from most cell types to date. We have shown that a standard curve and H2O2 assay can be created in cell growth media and will detect H2O2 leaving the cell down to approximately 1 pico mol levels in response to IgG and other ligands, drugs or receptor siRNA. The Fc receptor CD64 will be expressed on the surface of naive cells that will be fixed and used as a chromatography column to capture the receptor ligand IgG or Fc peptide (verses the control naive cells). A number of receptors for epidermal growth factors (EGF), interleukins (IL) and colony stimulating factors (CSF) and FLT3R were found with the IgG-Fc receptor complex. The FLT3 ligand was found to regulate the phagocytosis of IgG-coated microbeads and we will test the effect of the implicated in phagocytosis and Hydrogen peroxide production of IgG-coated microbeads. We will provide a new and powerful way to capture cell surface receptors and co-receptors, measure their roles in free radical release and phagocytosis, and then rapidly screen and test their protein ligands that might serve as biological drugs targeted against the receptor-specific inflammatory functions.