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

我们将通过质谱法和活细胞显微镜分析模型受体Fc受体在igg -微珠结合、识别和激活的时间过程。受体是在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，并针对它们制造新的特异性药物。许多对组织损伤的反应，如伤口和感染，涉及到一种叫做巨噬细胞的特殊细胞的激活，这种细胞反过来引发炎症反应。在大多数情况下，炎症是有益的，但偶尔异常的炎症反应会引发疾病过程，例如关节关节炎炎症，动脉粥样硬化性炎症，或者像癌症和阿尔茨海默氏痴呆症。人免疫球蛋白G （IgG）或其片段结晶（Fc）可标记入侵细菌进行破坏，可用于从巨噬细胞系（包括人分化的U937细胞和RAW巨噬细胞）中捕获其受体和/或共受体。我们的目标是激活并捕获非炎性的单价Fc γ受体- igg - s - s -生物素复合物，用于在链帕霉素上收集，并通过一步简单还原释放。聚集的igg微珠将激活并直接从活细胞表面捕获活化的吞噬复合物，用于质谱分离和表征。巨噬细胞包被配体的微球的质谱分析、生化分析和活细胞激光显微镜将揭示和证实Fc激活调节吞噬和自由基产生的机制。我们观察到Fc γ， epsilon, α， mu受体与Fc样受体（FCLR），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）的特异性亚型密切相关，这些受体在IgG-微珠上以超分子复合物的形式存在，而不是oxLDL或抗CD36微珠。对特异性发现的Fc受体和共受体复合物沉默RNA将证明共受体在吞噬作用或自由基产生中的作用。巨噬细胞和中性粒细胞是吞噬细胞，因此具有释放自由基的能力，如超氧化物（O2-*），在吞噬和破坏igg -细菌复合物过程中形成H2O2。然而，迄今为止，很少有绝对定量的方法可用于测量大多数细胞类型的低量H2O2产物。我们已经证明，可以在细胞生长介质中创建标准曲线和H2O2测定，并将检测出H2O2在IgG和其他配体、药物或受体siRNA的反应下离开细胞约1皮摩尔的水平。Fc受体CD64将在初始细胞表面表达，该细胞将被固定并用作色谱柱来捕获受体配体IgG或Fc肽（与对照初始细胞相比）。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）、集落刺激因子（CSF）和FLT3R受体。FLT3配体被发现调节igg包被微珠的吞噬作用，我们将测试其对igg包被微珠吞噬和过氧化氢产生的影响。我们将提供一种新的、强大的方法来捕获细胞表面受体和共受体，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，这些配体可能作为靶向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.