Chaperone-mediated heterotypic phase separation prevents the amyloid formation of the pathological Y145Stop variant of the prion protein

Lisha Arora, Dipankar Bhowmik, Snehasis Sarkar, Anusha Sarbahi, Sandeep K Rai, Samrat Mukhopadhyay
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Abstract

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.
伴侣介导的异型相分离可防止朊病毒蛋白的病理Y145Stop变体形成淀粉样蛋白
通过蛋白质和核酸的相分离形成的生物分子凝聚物对各种基本细胞功能的时空调节和细胞平衡的维持至关重要。然而,这种凝聚物异常的液固相变与几种致命的人类疾病有关。这种动态无膜区室可能含有一系列分子伴侣,它们可以调节参与形成这些生物凝聚物的蛋白质的相行为。在这里,我们发现热休克蛋白40(Hsp40)Ydj1具有抑制酶活性,它通过招募进入Y145Stop凝集物或通过Y145Stop-Ydj1双组分异型相分离防止Y145Stop构象转化为淀粉样纤维,从而增强与疾病相关的朊病毒蛋白终止密码子突变体(Y145Stop)的相分离。利用定点诱变、多色荧光成像、单液滴稳态和皮秒时间分辨荧光各向异性、光漂白后荧光恢复以及荧光相关光谱,我们描绘了支配 Y145Stop 和 Ydj1 异型相分离的复杂相互作用网络。我们还表明,这种异型凝聚物的性质可以通过促进多组分多相蛋白质-RNA 凝聚物形成的 RNA 进一步调整。我们的振动拉曼光谱结果与原子力显微镜成像相结合,揭示了 Ydj1 能有效地将 Y145Stop 的自组装转向动态抑制的非淀粉样蛋白生成途径,从而防止形成典型的淀粉样纤维。我们的发现强调了伴侣介导的异型相分离在调节与多种致命神经退行性疾病相关的异常相变和淀粉样蛋白形成方面的重要性。
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