Del Val Elba, Fernández-Vega Alejandro, Molina María, Cid Víctor J
Department of Microbiology and Parasitology, School of Pharmacy, Complutense University of Madrid, Pza. Ramón y Cajal s/n, Madrid, 28040, Spain.
Cell Mol Life Sci. 2025 Jul 25;82(1):288. doi: 10.1007/s00018-025-05827-1.
MyD88 is a key mediator of Toll-like receptor (TLR) signaling, orchestrating the innate immune response upon stimulation by pathogen-associated molecular patterns (PAMPs). Structurally, MyD88 consists of a Death domain (DD), a 20-amino acid N-terminal extension, and an intermediate (INT) region that connects it to a Toll/Interleukin-1 receptor (TIR) domain. At the core of the signaling complex known as myddosome, MyD88 undergoes homopolymeric interactions to propagate the signal. In this study, we use Saccharomyces cerevisiae as a heterologous model to assess the contribution of individual MyD88 domains to self-interaction and subcellular localization. In yeast, MyD88 localizes to endoplasmic reticulum-mitochondria encounter sites (ERMES). Here, we show that its DD is sufficient for attachment to the ERMES. Deletion of its 20 N-terminal residues increased MyD88 stability, shifting its aggregation pattern from patches to filaments. In contrast, a chimeric MyD88 variant bearing the plasma membrane-binding N-terminal extension of TIRAP, another TLR4-associated myddosome component, exhibited diffuse mitochondrial distribution. Moreover, we found that the ERMES-associated dynamin-like protein Dnm1, involved in mitochondrial fission, played a crucial role in MyD88 expression in yeast. On the other hand, the MyD88 TIR domain alone accumulated at lipid droplets in yeast, and its overexpression led to growth impairment and mitochondrial condensation. These findings suggest that MyD88 association with cellular membranes promotes self-assembly, a process essential for functional TLR signaling. Additionally, we adapted a tripartite GFP system to titrate MyD88 homopolymerization in yeast. Using this system, we observed that the oncogenic L252P mutation significantly reduced MyD88 ability to self-interact.
髓样分化因子88（MyD88）是Toll样受体（TLR）信号传导的关键介质，在病原体相关分子模式（PAMP）刺激后协调先天免疫反应。在结构上，MyD88由一个死亡结构域（DD）、一个20个氨基酸的N端延伸和一个将其连接到Toll/白细胞介素-1受体（TIR）结构域的中间（INT）区域组成。在被称为髓样分化因子88体（myddosome）的信号复合物的核心，MyD88经历同聚相互作用以传播信号。在本研究中，我们使用酿酒酵母作为异源模型来评估单个MyD88结构域对自身相互作用和亚细胞定位的贡献。在酵母中，MyD88定位于内质网-线粒体接触位点（ERMES）。在此，我们表明其DD足以附着于ERMES。删除其20个N端残基可增加MyD88的稳定性，将其聚集模式从斑块转变为细丝。相反，携带另一种TLR4相关髓样分化因子88体成分TIRAP的质膜结合N端延伸的嵌合MyD88变体表现出线粒体的弥漫性分布。此外，我们发现参与线粒体分裂的与ERMES相关的动力蛋白样蛋白Dnm1在酵母中MyD88的表达中起关键作用。另一方面，单独的MyD88 TIR结构域在酵母的脂滴处积累，其过表达导致生长受损和线粒体凝聚。这些发现表明MyD88与细胞膜的结合促进了自组装，这是功能性TLR信号传导所必需的过程。此外，我们采用了一种三方绿色荧光蛋白（GFP）系统来滴定酵母中MyD88的同聚作用。使用该系统，我们观察到致癌性L252P突变显著降低了MyD88自身相互作用的能力。
