| Detection of Apoptosis-like Cell Death in Ustilago maydis by Annexin V-FITC Staining
|
|
Author:
Date:
2018-08-05
[Abstract] Programmed cell death (PCD) guides the transition between key developmental stages in many organisms. PCD also remains an important fate for many organisms upon exposure to different stress conditions. Therefore, an insight into the progression of PCD during the execution of a biological phenomenon can yield significant details of the underlying mechanism. Apoptosis, as well as apoptosis-like programmed cell death, constitutes one of the forms of PCD in higher and lower eukaryotes respectively. Flipping of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet is among the different hallmarks of apoptosis/apoptosis-like PCD that marks the initiation of the said cell death event. This flipping can be detected through staining of the target cells using ...
[摘要] 程序性细胞死亡（PCD）指导许多生物体的关键发育阶段之间的过渡。 PCD在暴露于不同的胁迫条件下仍然是许多生物的重要命运。因此，在执行生物现象期间洞察PCD的进展可以产生潜在机制的重要细节。细胞凋亡以及凋亡样程序性细胞死亡分别构成高等和低等真核生物中PCD的一种形式。将磷脂酰丝氨酸（PS）从质膜的内部小叶翻转到外部小叶是凋亡/凋亡样PCD的不同标志之一，其标志着所述细胞死亡事件的开始。可以使用与PS特异性结合的膜联蛋白V-FITC通过染色靶细胞来检测这种翻转。在 Ustilago maydis 中，由于细胞壁的存在，膜联蛋白V-FITC对外露PS的染色是困难的。因此，这种染色的关键在于，在不显着改变下面的质膜结构/拓扑结构的情况下，温和地去除细胞壁。该协议强调了PS染色对 Ustilago maydis 中应激细胞原生质体的依赖性。
【背景】PS外化是早期可以检测到的凋亡样PCD的标志之一（Martin et al。，1995）。因此，质膜在细胞外膜上的出现标志着凋亡细胞死亡现象的发生。 Ustilago maydis 是一种生物营养植物病原体并感染寄主植物 Zea mays 。 U的生命周期。已证明maydis ...
|
|
|
| An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
|
|
Author:
Date:
2017-11-20
[Abstract] We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et ...
[摘要] 我们最近报道了一种针对内源性感兴趣蛋白（POI）的靶向蛋白水解的亲和指导PROtein导弹（AdPROM）系统（Fulcher等人，2016和2017）。 AdPROM由Von Hippel Lindau（VHL）蛋白组成，Cullin 2 E3连接酶底物受体（Bosu and Kipreos，2008），与识别细胞中靶蛋白的高亲和力多肽结合剂（如骆驼科纳米抗体）缀合。当在细胞中引入时，靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集，我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体（Sharm等人，2013; Fridy等人。，2014; Schmidt et al。，2016）。在此协议中，我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征，并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性，这些方法需要长时间的治疗并与脱靶效应相关联，而CRISPR / Cas9基因编辑并不总是可行的。
【背景】该协议使人们能够在哺乳动物细胞系中设计，构建和表达AdPROM VHL-nano ...
|
|
|
| Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
|
|
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。 ...
|
|
|