Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation.
Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation.
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DOI：
10.1371/journal.pgen.1006708
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发表时间：
2017-04
期刊：
影响因子：
4.5
通讯作者：
Grant CM
中科院分区：
文献类型：
--
作者：
Speldewinde SH;Doronina VA;Tuite MF;Grant CM
哺乳动物和真菌的朊病毒从头开始出现，然而，其机制在分子方面知之甚少。一个很大的可能性是，对非朊病毒形式的蛋白质的氧化损伤可能是影响其可遗传朊病毒构象形成的重要触发因素。我们已经研究了氧化应激诱导的酵母[PSI+]朊病毒的形成，这是Sup35翻译终止因子的构象改变。我们使用串联亲和纯化（TAP）和质谱法来鉴定与Tsa1 Tsa2抗氧化突变体中Sup35相关的蛋白质，以解决Sup35在氧化应激条件下形成[PSI+]朊病毒的机制。这项分析确定了几个组成部分的皮质肌动蛋白细胞骨架，包括ABP1肌动蛋白成核促进因子，我们发现，ABP1基因的缺失废除氧化剂诱导的[PSI+]朊病毒的形成。自发[PSI+]朊病毒形成的频率可以通过Sup35的过表达而增加，因为过量的Sup35增加了形成朊病毒种子的可能性。与氧化剂诱导的[PSI+]朊病毒形成相反，过度表达诱导的[PSI+]朊病毒形成在abp1突变体中仅受到适度影响。此外，用latrunculin A处理酵母细胞以破坏肌动蛋白电缆和补丁的形成，废除了氧化剂诱导的，但不是过度表达诱导的[PSI+]朊病毒形成，表明朊病毒形成的机制差异。[PIN+]，Rnq 1的朊病毒形式，定位于IPOD（不溶性蛋白质存款），并被认为影响其他蛋白质的聚集。我们发现Sup35在氧化应激条件下被氧化和聚集，但不与Rnq1在abp1突变体中共定位，这可能是[PSI+]朊病毒形成频率降低的原因。朊病毒是由错误折叠的蛋白质组成的感染因子，并且与进行性神经退行性疾病如克雅氏病（CJD）有关。大多数朊病毒疾病偶发发生，然后通过诱导蛋白质错误折叠以仅蛋白质机制传播。目前很少有人知道正常可溶性蛋白质如何自发形成其朊病毒形式。以前的研究已经暗示了某些蛋白质的非朊病毒形式的氧化损伤是形成其可遗传朊病毒构象的重要触发因素。使用酵母朊病毒模型，我们发现，皮质肌动蛋白细胞骨架是所需的氧化蛋白质的转变，其遗传感染性构象。在破坏皮质肌动蛋白细胞骨架的突变体中，氧化蛋白聚集，但不定位于其正常的淀粉样蛋白沉积位点，称为IPOD。IPOD作为一个网站，朊病毒蛋白进行片段化和播种，我们表明，防止肌动蛋白介导的本地化到这个网站防止自发和氧化剂诱导的朊病毒形成。
Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation. Prions are infectious agents which are composed of misfolded proteins and have been implicated in progressive neurodegenerative diseases such as Creutzfeldt Jakob Disease (CJD). Most prion diseases occur sporadically and are then propagated in a protein-only mechanism via induced protein misfolding. Little is currently known regarding how normally soluble proteins spontaneously form their prion forms. Previous studies have implicated oxidative damage of the non-prion form of some proteins as an important trigger for the formation of their heritable prion conformation. Using a yeast prion model we found that the cortical actin cytoskeleton is required for the transition of an oxidized protein to its heritable infectious conformation. In mutants which disrupt the cortical actin cytoskeleton, the oxidized protein aggregates, but does not localize to its normal amyloid deposition site, termed the IPOD. The IPOD serves as a site where prion proteins undergo fragmentation and seeding and we show that preventing actin-mediated localization to this site prevents both spontaneous and oxidant-induced prion formation.