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

用于分析和靶向 Fc 受体超分子复合物的生物物理和生化技术

• 批准号: RGPIN-2014-03967
• 负责人: Marshall, John
• 金额: $ 2.55万
• 依托单位: Ryerson University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587521
• 关键词: Biophysical; biochemical; techniques; analysis; targeting

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.

我们将通过质谱仪和活细胞显微镜分析一个模型受体，即Fc受体，分析免疫球蛋白与微球结合、识别和激活的时间过程。受体是细胞表面表达的蛋白质，控制着对体内循环信使的反应，是治疗疾病和疼痛的关键靶点。人们非常需要了解和比较细胞表面的受体，并创造出针对它们的新的和特定的药物。对组织损伤的许多反应，如创伤和感染，都涉及到被称为巨噬细胞的特殊细胞的激活，而巨噬细胞反过来又触发了炎症反应。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、动脉粥样硬化性炎症或癌症和阿尔茨海默氏症。人免疫球蛋白G(Ig G)或其可结晶片段(Fc)标志着入侵细菌的破坏，可用于从巨噬细胞系(包括人分化的U937细胞和原始巨噬细胞)捕获它们的受体和/或辅助受体。我们的目标是激活并捕获非炎症性的单价Fcγ受体-Ig G-S-S-生物素复合体，用于在链霉亲和素上收集，并通过简单的一步还原释放。聚集的免疫球蛋白微球将激活并直接从活细胞表面捕获激活的吞噬复合体，用于分离和质谱分析。将配体包裹的微球与培养的巨噬细胞结合，进行质谱分析、生化分析和活细胞激光显微镜观察，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Fcγ、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)的特定亚型密切相关，这些受体共同存在于Ig G-微球上的超分子复合体中，而不是oxLDL或抗CD36微球。沉默针对特定发现的Fc受体和辅助受体的复合体的RNA将证明辅助受体在吞噬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-*)，在吞噬和破坏免疫球蛋白-细菌复合体的过程中会导致过氧化氢的形成。然而，到目前为止，几乎没有绝对定量的方法来测量大多数细胞类型的低量过氧化氢产物。我们已经证明，可以在细胞生长介质中创建标准曲线和H_2O_2检测，并将检测到H_2O_2，使细胞对免疫球蛋白和其他配体、药物或受体siRNA的反应降至约1皮摩尔水平。Fc受体CD64将被表达在初始细胞的表面，该表面将被固定并用作层析柱以捕获受体配体Ig G或Fc肽(与对照初始细胞相反)。Ig G-Fc受体复合体具有多种表皮生长因子(EGF)、白细胞介素2(IL)、集落刺激因子(CSF)和Flt3R受体。我们发现Flt3配体可以调节包被免疫球蛋白的微球的吞噬功能，我们将测试该配体对包被免疫球蛋白的微球的吞噬和过氧化氢产生的影响。我们将提供一种新的有效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配体，这些配体可能作为针对受体特异性炎症功能的生物药物。