隔离增效变种的Hsp104利用酵母Proteinopathy模型
Summary
Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.
Abstract
Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson’s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.
Introduction
酵母proteinopathy模型已经开发了用于蛋白质错误折叠疾病，包括肌萎缩侧索硬化症（ALS）和帕金森氏病（PD）1-3。蛋白质TDP-43和FUS，在ALS患者其错误折叠的表达，是有毒的，mislocalize形成细胞质聚集在酵母1,2。类似地，α突触核蛋白（α-顺式），其牵涉在PD的表达，是有毒的和mislocalizes形成细胞质聚集体在酵母3。这些特性概括在表型的患者患有这些疾病4,5。因此，酵母模型提供了一个有益的平台，筛选，以防止或逆转这些表型2,6-13蛋白质或小分子。我们感兴趣的蛋白，其能够可逆聚集和毒性由于TDP-43，FUS，和α – 顺式的发展。我们注重的Hsp104，一个AAA +蛋白酵母是唯一能解聚蛋白的两个FROM无定形聚集和淀粉样蛋白在酵母中，但它没有人类同源14,15。的Hsp104被精细地调谐到分解的内源性酵母朊病毒，并具有唯一的解聚与人类神经变性疾病的底物，它从不通常遇到16,17的能力有限。因此，我们的目标是工程师的Hsp104的增强版本，它们能够efficaciously分解这些人的基板。要做到这一点，我们构建大库使用易错PCR变种的Hsp104;这些库可以利用酵母proteinopathy 17款进行筛选。我们采用了域针对性的方法来构建和筛选库，为的Hsp104是非常大的17。我们最初集中在中间域的Hsp104 17的纵向（MD），但类似的方法可以用于筛选其他领域。这些模型可以筛选disaggregase活动直接，而不是如表面显示，这是剩下的替代技术ricted用于监测结合18。
我们的协议是基于两个筛选步骤（ 图1）。首先，抑制酵母病衬底的毒性的Hsp104变体被选择。这样做，所述变体的Hsp104和疾病相关的衬底被共转化进ΔHSP104酵母。我们采用ΔHSP104酵母探索在没有野生型（WT）17的Hsp104的Hsp104序列空间。重要的是，缺失的Hsp104不影响α-顺，FUS，或TDP-43的毒性在酵母和的Hsp104 WT的表达提供了最小的救援1,13,17。酵母，然后铺于诱导培养基来诱导这两种蛋白质的表达。酵母窝藏的压制殖民地的疾病相关基板胙增长毒性变异的Hsp104。这些变体被选择用于进一步分析，而菌落保持变体不抑制毒性死亡。不过，误报是在该屏幕一个很大的问题。 TDP-43，FUS，和α – 顺式的表达是高度有毒的，它创建了对于毒性无关的Hsp104变体的自发遗传抑制器的外观强烈的选择压力被表达。因此，我们已经使用了一个辅助屏幕也相对高吞吐量，以消除这些非特异性毒性抑制器17。在此二次筛选，选择的酵母与5- Fluorootic酸（5-FOA）处理以对抗选择为的Hsp104质粒19。的菌株，然后通过点样测定，以确保所述衬底的毒性的Hsp104质粒丢失后恢复评估衬底（TDP-43，FUS，或α – 顺式）的毒性。因此，酵母，其中毒性在本二次筛选恢复推测最初显示毒性抑制因的Hsp104变体的存在。这些酵母被指定为“命中”，然后将质粒的Hsp104应回收并测序，以确定在所述的Hsp104基因17（ 图1）中的突变。任何命中应该然后通过构建突变独立地使用定点诱变，然后再测试的毒性抑制作确认。该协议的潜在应用是广泛的。使用这些方法，任何类型的蛋白质的文库可以进行筛选，即抑制任何底物蛋白质是在酵母中的有毒的毒性变异体。
Protocol
Representative Results
Discussion
在这里，我们提出我们的方法来隔离的抑制用酵母proteinopathy车型疾病相关基材的毒性增效的Hsp104变种。使用这种方法，变体大文库可以筛选在高吞吐量，唯一的限制是对所有通过5-FOA二次筛选变体的数量。通过在96孔格式这些步骤，我们例行公事地检查了200个命中在5-FOA步过1-2周的课程时间。在最初的筛选步骤中求出的匹配个数将变化很大程度上取决于衬底毒性和每一特定库的组成。当测序命中，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director’s New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).
Materials
| Table of Specific Materials/Equipment | |||
| Name | Company | Catalog Number | Comments |
| GeneMorphII EZClone Domain Mutagenesis Kit | Agilent | 200552 | |
| 150mm Petri dishes | Falcon | 351058 | |
| 5-Fluorootic Acid | Research Products International | f10501-5.0 | |
| 96-DeepWell 2mL Plates | Eppendorf | 0030 502.302 | |
| 96 bold replicator tool | V&P Scientific | vp-404 | |
| ExoSAP-IT | Affymetrix | 78200 |
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