| 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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| Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies
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Author:
Date:
2016-11-20
[Abstract] The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then isolated through culturing, filtering and magnetic separation using cell-surface antigen markers. Here we provide a comprehensive protocol on how to isolate and culture individual cell types from whole endometrial tissues for use in in vitro experiments.
[摘要] 从人子宫内膜活组织检查中分离和原代培养细胞为生殖和妇科研究提供了有价值的实验材料。从同意的妇女收集完整的子宫内膜活检组织并用胶原酶和DNA酶I消化以从细胞外基质中分离细胞。然后通过使用细胞表面抗原标记物的培养，过滤和磁性分离来分离细胞群。在这里，我们提供了如何从整个子宫内膜组织分离和培养个体细胞类型以用于体外实验的全面方案。
[背景] 人子宫内膜是子宫内层最粘的层。其由柱状上皮和基础基质层组成，其经历循环再生，生长和响应于循环激素的转化。子宫内膜衬里分化为腺分泌表型为囊胚植入和成功怀孕提供了一个好客的环境。在没有怀孕这个层脱落，导致月经。从人子宫内膜活组织检查中分离和培养细胞允许在体外功能评估和与患者结果相关的细胞特征的研究。子宫内膜细胞的分离和培养是研究妇科和产科医学的许多方面的非常宝贵的研究模型，包括不孕，植入失败，复发性流产和月经紊乱。全人子宫内膜活检包含人子宫内膜基质细胞（HESCs），腔和子宫内膜上皮细胞（HEEC），红细胞和免疫细胞的混合群体。 ...
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| Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1
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Author:
Date:
2014-04-05
[Abstract] The fate of mRNA, in particular its stability, localization and rate of translation is regulated by RNA binding proteins assembling to messenger ribonucleoprotein (mRNP) complexes. To investigate the transcriptome-wide RNA binding sites of UPF1, the core factor of nonsense-mediated mRNA decay (NMD), we performed individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) (Zund et al., 2013) followed by high-throughput sequencing. The presented protocol is optimized to investigate the RNA-binding sites of UPF1 and is based on previously described studies (Konig et al., 2010; Konig et al., 2011; Hafner et al., 2010). We want to thank the Group of Mihaela Zavolan (Swiss Institute of Bioinformatics, Basel, Switzerland) and Jernej Ule ...
[摘要] mRNA的命运，特别是其稳定性，定位和翻译速率由装配到信使核糖核蛋白（mRNP）复合物的RNA结合蛋白调节。 为了研究无义介导的mRNA衰变（NMD）的核心因子UPF1的转录组范围的RNA结合位点，我们进行单个核苷酸分辨率的UV交联和免疫沉淀（iCLIP）（Zund等，/em>，2013），然后进行高通量测序。 优化所提出的方案以研究UPF1的RNA结合位点并且基于先前描述的研究（Konig等人，2010; Konig等人，2011; Hafner等人，2010）。 我们要感谢Mihaela Zavolan（瑞士巴塞尔瑞士生物信息学研究所）和Jernej Ule（英国剑桥的分子生物学医学研究委员会实验室）组织这些实验的技术支持。
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