Department of Dermatology, Inselspital Bern University Hospital, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Department of Dermatology, Inselspital Bern University Hospital, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
J Invest Dermatol. 2014 Nov;134(11):2776-2783. doi: 10.1038/jid.2014.255. Epub 2014 Jun 18.
Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.
桥粒斑蛋白是斑联蛋白家族中的细胞连接蛋白，将由角蛋白 5 和 14（K5/K14）组成的中间丝（IF）网络锚定到半桥粒中，半桥粒是基底层角质形成细胞中的连接黏附复合体。这些蛋白的遗传改变导致单纯型大疱性表皮松解症（EBS），其特征是细胞结构和细胞脆性紊乱。位于已记录的桥粒斑蛋白 IF 结合位点（由斑联蛋白重复结构域（PRD）B5 和连接子组成）之后的突变，以及 K5 或 K14 中的突变如何导致 EBS 的机制尚不清楚。我们使用不同的方法研究了桥粒斑蛋白 C 末端（包含四个结构域，即 PRD B5、连接子、PRD C 和 C 末端）与 K5/K14 的相互作用，包括一种基于增强型绿色荧光蛋白标记蛋白（FluoBACE）的快速灵敏的荧光蛋白结合测定法。我们的结果表明，桥粒斑蛋白 C 末端的所有四个结构域都有助于其与 K5/K14 的结合，并协同作用以确保有效的 IF 结合。与单体角蛋白或 IF 组装中间体相比，桥粒斑蛋白 C 末端主要与 K5/K14 的 coil 1 结构域相互作用，并与 K5/K14 细丝结合更广泛。这些发现表明桥粒斑蛋白与 K5/K14 细丝的多模块化结合，并深入了解与桥粒斑蛋白、K5 或 K14 基因中的致病性突变相关的 EBS 的分子基础。
