| An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
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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 ...
[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 ...
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| Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay
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Author:
Date:
2016-08-20
[Abstract] Cell-cell adhesion ensures tight contacts between neighbouring cells, which is necessary for cell segregation, as well as for the morphological and functional differentiation of different tissues. Evidently there are cell-cell recognition systems that make cells of the same type preferentially adherent to one another. Homotypic cell adhesion is particularly important in mediating a range of physiological processes such as cell survival, migration and remodeling of vessels. Thus in the present study we selected two populations of endothelial progenitor cells which are from the same donor to ...
[摘要] 细胞粘附保证了相邻细胞之间的紧密接触，这对于细胞分离以及不同组织的形态和功能分化是必要的。显然，存在使相同类型的细胞优先彼此粘附的细胞 - 细胞识别系统。同型细胞粘附在介导一系列生理过程例如细胞存活，迁移和血管重塑中特别重要。因此，在本研究中，我们选择两个来自相同供体的内皮祖细胞群体，以研究小分子化合物Icariin对同型细胞粘附的可能影响。许多血管生成因子可以破坏细胞间连接的组织，导致内皮屏障开放。在本研究中，我们观察到Icariin治疗减少EPC中VE-钙粘蛋白表达的水平，表明细胞 - 细胞粘附减少 - 证明Icariin的促血管生成作用。总之，所观察到的EPCs的同型粘附的丧失可能有助于由Icariin施加的增强的血管生成作用。
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