EMBL Australia Node in Single Molecule Science, University of New South Wales, Kensington, NSW, 2052, Australia.
Institute for Molecular Bioscience, University of Queensland, QLD, Brisbane, 4072, Australia.
BMC Biol. 2018 Dec 24;16(1):149. doi: 10.1186/s12915-018-0611-7.
Higher-order self-assembly of proteins, or "prion-like" polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level.
We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88.
We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital "all-or-none" responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.
蛋白质的高级自组装，或“类朊病毒”聚合，现在作为一种简单而强大的信号放大机制出现，特别是在先天免疫系统中，病原体或危险相关分子模式的识别需要在细胞内引发强烈的二元反应。MyD88 是 TLR 下游的一种重要衔接蛋白，是参与高级自组装信号的最新候选蛋白之一。在这种新视角下，我们着手重新解释聚合在 MyD88 相关疾病中的作用，并在分子水平上研究疾病相关点突变 L93P、R196C 和 L252P/L265P 的影响。
我们首先开发了新的体外策略来描述生理水平下聚合全长 MyD88 的行为。为此，我们使用了单分子荧光波动光谱学，结合真核细胞无细胞蛋白表达系统。然后，我们能够在低蛋白浓度和无需纯化的情况下探索全长 MyD88 的聚合倾向，并将其与已证明具有自身组装特性的分离 TIR 结构域和死亡结构域的行为进行比较。这些实验表明，两个结构域的存在是协同导致蛋白质有效聚合所必需的。然后，我们对 MyD88 的三个病理突变体进行了表征。
我们发现所有突变都阻止了 MyD88 完全聚合的能力。有趣的是，我们表明与 L93P 和 R196C 相反，L252P 是一种功能获得性突变，它允许 MyD88 突变体形成极其稳定的寡聚物，即使在低纳摩尔浓度下也是如此。因此，我们的结果为 MyD88 形成的“数字全或无”反应和 MyD88 的致癌突变的行为提供了新的认识。
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