| Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
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Author:
Date:
2017-08-20
[Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough ...
[摘要] 我们已经开发了产生组氨酸激酶DesK及其同源反应调节物DesR的位点特异性变体的方案，有助于捕获蛋白质的不同信号状态。两个合作伙伴在大肠杆菌中的共表达，确保调节剂过量，对于DesK：DesR复合物的可溶性生产和进一步纯化是至关重要的。通过使用分子置换的X射线晶体学解决了捕获在磷酸转移酶和磷酸酶反应步骤中的复合物的3D结构。该解决方案不是微不足道的，我们发现在用作搜索探针的硅片生成的模型中，有助于将大部分复合物放置在不对称单元中。电子密度图就足够清楚了，可以进行人工建模，获得完整的原子模型。这些方法有助于解决细菌信号领域的主要挑战，即获得稳定的激酶：调节复合物，具有不同的构象状态，适用于高分辨率晶体学研究。
【背景】关于细菌信号复合物，特别是双组分系统（TCS）的结构信息仍然很少（Casino et al。，2009; Gao and Stock，2009）。 TCS包含几乎所有细菌中的感觉组氨酸激酶（HK）和响应调节剂（RR）配偶体，它们允许细胞感知环境并通过适应性反应相应地反应。尽管在信号传输中这种切换机制的重要性（Trajtenberg等，2016），结构信息对于采用不同功能状态的TCS复合体甚至更为有限。我们研究了DesK-DesR途径（de Mendoza，2014），一种来自枯草芽孢杆菌的TCS，其参与调节细胞膜组成以适应降低双层流动性的线索，如冷休克。 ...
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| Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
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Author:
Date:
2015-06-05
[Abstract] Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with ...
[摘要] 酪氨酸，丝氨酸和苏氨酸残基的磷酸化对于控制参与各种细胞事件的蛋白质活性是至关重要的。各种激酶和磷酸酶调节许多不同细胞信号传导途径中的细胞内蛋白磷酸化。这些途径包括T和B细胞信号传导，调节生长和细胞周期控制，加上细胞因子，趋化因子和应激反应。 Phosphoflow测定结合磷蛋白特异性抗体与流式细胞术的力量，以增强磷蛋白研究。在我们的测定中，外周血单核细胞被细胞因子刺激，固定，用用MAXPAR（商品名）金属螯合聚合物标记并用甲醇透化的抗体的混合物表面染色。然后用细胞内磷酸特异性抗体染色。
我们使用CyTOF TM 质谱仪获取ICP-MS（电感耦合等离子体质谱）数据。选择的当前质量窗口大约是AW 103-203，其包括用于大多数抗体标记的镧系元素，以及用于DNA嵌入剂的铱和铑。使用FlowJo软件的双计数信号数据的后续分析允许基于每个质量通道中的双计数信号来分析细胞类型。确定每种细胞类型的百分比，并报告为父细胞类型的百分比。报道中值以定量响应刺激的每种蛋白质的磷酸化水平。比较样品之间的磷酸化水平可以提供对免疫系统状态的了解。
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| Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
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Author:
Date:
2015-04-05
[Abstract] Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also be used in combination with other flow cytometry protocols for ...
[摘要] 细胞因子的产生在免疫应答中起重要作用。细胞因子参与许多不同的途径，包括通过IFNγ诱导许多抗病毒蛋白，通过IL-2诱导T细胞增殖以及抑制病毒基因表达和TNFα的复制。细胞因子不是预先形成的因子，但是响应于细胞激活而快速产生和分泌。通过流式细胞术的细胞内细胞因子检测已经成为研究单细胞水平的细胞因子产生的首要技术。它检测细胞刺激后细胞因子在内质网内的产生和积累，允许直接TH1对TH2测定。它也可以与其他流式细胞术协议结合使用细胞表面标记或与MHC多聚体进行免疫分型，以检测抗原特异性反应，使其成为一种非常灵活和通用的方法。这种能力，结合仪器的高通量性质，使细胞内细胞因子染色与现有的单细胞技术如ELISPOT，有限稀释和T细胞克隆相比具有巨大的优势。细胞内细胞因子染色的主要步骤如下：
1。使用特异性肽或非特异性活化混合物将细胞活化几小时;
2。加入蛋白质转运抑制剂（例如布雷菲德菌素A）以将细胞因子保留在细胞内;
。接下来，加入EDTA以从活化容器中除去粘附细胞;
4。洗涤后，可将细胞表面标记的抗体加入细胞中;
5。然后将细胞固定在多聚甲醛中并透化;
6。加入抗细胞因子抗体，并且可以通过流式细胞仪分析细胞。
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