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.).

1.生成库<ol><li>构造的Hsp104利用特定于域的易错PCR文库，首先扩增感兴趣与易出错的DNA聚合酶20的域。 </li><li>纯化PCR产物通过凝胶提取。 </li><li>使用标准的定点诱变协议进行大引物延伸步骤:结合为50ng模板质粒，250ng的大引物，200μMdNTPs和高保真DNA聚合酶在PCR缓冲液，并稀释至50μl总体积PCR级水20 。运行一个标准的PCR程序。 注意:用于将基于要被扩增的基因的特定区域而变化的特定的引物。 <ol><li>下述PCR，在37℃下消化亲代DNA模板与1μl的DPN限制性内切酶处理2小时。 </li></ol></li><li>通过电穿孔或其他方法在ultracompetent 大肠杆菌转化库大肠杆菌和小量净化它。 注:库代差异极大基于给定实验的目的。的Hsp104是一个非常大的蛋白质，因此它是不现实的随机化的完整基因，这就是为什么我们利用特定于域的易错PCR。此外，的Hsp104的结构仍然知之甚少，使得定向库挑战21的设计。库可以用定向或随机的方法来诱变来构建，唯一的限制是，该模板质粒骨架应包含URA3基因，以允许在葡萄糖介质5-FOA反选择。 </li></ol> 2.转型的Hsp104图书馆<ol><li>使用标准的醋酸锂/ PEG转化协议22集成的疾病相关的基板为W303aΔHSP104酵母。选择单菌落，筛选它们的毒性隔离关联基板23该病的高毒性菌株。 注:使用集成的应变和IsolaTE的一种单菌落，以确保在所有细胞中表达的平等。克隆病基底到质粒允许整合下比尿嘧啶以外的任何标记（我们使用组氨酸）和所述的Hsp104文库的基因导入pAG416GAL质粒（尿嘧啶标记）24。为了让5-FOA反选择，确保库是从与尿嘧啶标记的质粒表达。其他的酵母菌株也可使用。我们注意到使用W303a和BY4741酵母菌株在WT和ΔHSP104背景（MEJ和JS，未发表意见）相似的毒性抑制的表型。 </li><li>使用相同的醋酸锂/ PEG转化协议22改造图书馆的Hsp104这个压力。向上扩展变换适当地保持库的序列空间和保存预测的文库大小。 </li><li>制版用足够板的转化混合物到非诱导，选择性平板（ 例如 SD-他浦）确保大量菌落的生长。使用大陪替氏培养皿（150毫米），最小化所需的板的数量。回收利用板的转化使转化效率进行评估。 </li><li>变换的Hsp104 WT和载体的阴性对照平行。 </li></ol> 3.筛选抑制Proteotoxicity的<ol><li>利用棉子糖补充丢失的介质（ 如 SRaff，他的浦）洗掉板的殖民地。用血清学移液器和无菌敷料木从板松开殖民地。转移的液体洗涤至50ml锥形管中，涡旋彻底分离的细胞中的任何团块。稀释至轻微浑浊的文化，外径600〜0.025。 注:这里的棉子糖的作用是缓解诱导葡萄糖介导的抑制的细胞，从而引发细胞的半乳糖介导的诱导。 </li><li>振荡在3一夜长大稀释文化，棉子糖辍学媒体0ºC。成长WT的Hsp104和矢量控制并行。 </li><li>第二天早晨，制版浓度范围到个别半乳糖（ 例如 SGal-他浦）的板（1微升至2毫升浓培养于400μl总体积）。镀了广泛的浓度范围内可以保证一板将具有单个菌落。所需的实际浓度取决于所关注的疾病衬底的毒性。 </li><li>归一化矢量和的Hsp104 野生型培养物到库中的OD 600和板等体积的比较相对于所述控件库的增长。 </li><li>为了评估选择严格，板块葡萄糖库（抑制）的媒体。孵育平板2-3天，在30°C以上，直至菌落出现（ 图2）。评估所产生的菌落并比较对照。 注:通常大型和小型的殖民地获得，但菌落大小和行为之间无相关性ivity观察。筛选使用5-FOA计数器选择技术（步骤4），以尽量减少结转测序误报这些潜在的命中。 </li></ol> 4. 5-FOA反选择和定位，以消除误报<ol><li>连胜出单菌落重复到双人和单人辍学介质（ 如 SD-他的浦和SD-他的车牌），并在30ºC成长过夜。重复使用矢量和WT的Hsp104控制。镀上的SD-他之前的5-FOA，增加的可能性，细胞就会失去了质粒的Hsp104当它们被镀上5-FOA增加了5-FOA步骤的效率。 </li><li>为了防止板变干，敷SD-他的浦板块的​​封口膜，并储存在4ºC，同时进行5-FOA屏幕。这些板最终将被用于测序。 </li><li>条纹从菌落中的SD-他的板，以单菌落对5-FOA平板（YPD培养基+1克/ L的5-FOA）并孵育1-2天，在30＆＃186; C，直到单菌落出现。这里，5-FOA转化成毒性产物（5-氟脱氧尿苷）在包含URA3基因的细胞。因此，任何细胞仍窝藏质粒的Hsp104会死。 </li><li>连胜从5-FOA平板式两份的单菌落到SD浦和SD-他的板。测试3个菌落为每个命中的可能性增加，至少一个集落的每个命中会失去了的Hsp104质粒。孵育1-2天，在30℃下。已经失去了质粒的Hsp104将生长在SD-他的板，但没有SD浦板块的殖民地。 </li><li>成长已经失去了质粒的Hsp104用于探测试验菌株。长株棉子糖辍学媒体饱和度（ 如 SRaff-他）在96深孔2毫升板过夜30ºC震荡。一定要包括5-FOA处理的对照菌株。 </li><li>准备板的斑点检测。使用多道移液器，分装200微升棉子糖辍学介质（ 如 SRaff-他）每以及一个96孔板的，保留列1和7为纯的培养物。等份加入250μl各16的饱和培养物的成列1和板7。 </li><li>连续稀释的培养物5倍到板的各列中，用多通道移液管充分混合。从柱6和12去除50微升稀释的培养物，以确保每个孔中的最终体积为200微升。这是必要的，以确保均匀的斑点。 </li><li>用96螺栓复制工具（青蛙），发现一式两份的培养到SD-他和SGal，他的板。孵育所述板在30°C以上为2-3天。 </li><li>选择用于测序显示出对SGal-他板类似于对照的毒性菌株。放弃误报菌株，但并不像控制有毒。 5-FOA也是对自己往往有毒的，所以放弃，表现出对SD-他的片生长不良或表现出显着毒性较大比单独的疾病基板株（ <strong&gt;的图3）。 </li></ol> 5.测序的变体的Hsp104菌落PCR <ol><li>从SD-他浦板刮〜10微升酵母在3微升的20mM的NaOH中的PCR条管和冻融裂解细胞。放置在-80ºC冷冻带状管10分钟，或在液氮中，然后孵育99ºC在热循环仪进行10分钟。稀释该菌株以100微升用PCR级水。 </li><li>准备PCR反应:5μLPCR模板，0.5微升100μM的引物各0.5微升10毫的dNTP，在PCR缓冲液0.25微升的DNA聚合酶，并进行多达25微升总体积PCR级水。设计引物来扩增随机区域加上两端约100bp。最小化的放大区域的尺寸，以增加成功的扩增的可能性。运行一个标准的PCR程序。 </li><li>分析通过琼脂糖凝胶电泳的样品，以确认适当的SIZ的PCR产物的扩增Ë。重复PCR，如果没有产品存在。 注:PCR失败通常是由于过多的酵母在步骤5.1被使用。 </li><li>制备样品用于DNA测序。除去PCR引物或者通过PCR纯化或使用PCR产物的清除剂，如外切核酸酶I和虾碱性磷酸酶的酶（ExoSAP-IT）的降解单链DNA。此试剂酶促降解未掺入的引物和dNTP，从而允许更高的吞吐量。 </li><li>序列的PCR产物。一定要深入分析测序图谱的基因突变。突变可以被观察到的两个核苷酸在给定的位点（ 图4）的混合物，如酵母经常怀有多种质粒。 </li></ol>

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The authors have nothing to disclose.

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，就必须仔细地分析所有的测序色谱图，因为质粒的混合物，典型地存在于每个细胞中。 我们有时注意到的Hsp104 WT的大百分比被分离为"命中"。这可能是由于自发的基因的存在抑制器或非常弱的活动。为了解决这个问题，我们已发现，筛选在升高的温度（ 例如 34℃），能减少&#39;命中&#39;，它的Hsp104 WT中的百分比。此外，还必须重新克隆任何测序命中独立评估的任何新颖的变体的活性，并确保质粒的纯度。一旦新的命中被确定，它们可以被评估为在酵母中抑制聚合的和生物化学特征。 我们已经使用了这些方法，分离增效的Hsp104变体针对TDP-43，FUS，α突触核蛋白，并使用纯蛋白生化测定17证实它们的活性。最终，这些方法可以被用于筛选蛋白质文库针对任何感兴趣的底物是在酵母中的毒性。

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。

<table><tbody><tr><td>GeneMorphII EZClone Domain Mutagenesis Kit</td><td>Agilent</td><td>200552</td><td></td></tr><tr><td>150 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>5-Fluorootic Acid</td><td>Research Products International</td><td>f10501-5.0</td><td></td></tr><tr><td>96-DeepWell 2 ml Plates</td><td>Eppendorf</td><td>0030 502.302</td><td></td></tr><tr><td>96 bold replicator tool</td><td>V&amp;amp;P Scientific</td><td>vp-404</td><td></td></tr><tr><td>ExoSAP-IT</td><td>Affymetrix</td><td>78200</td><td></td></tr></tbody></table>

isolating potentiated hsp104 variants using yeast proteinopathy models

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 &#945;-synuclein, implicated in Parkinson&#8217;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 &#945;-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.

我们已经构建了随机的中间领域的Hsp104变体库，并筛选其用于抑制TDP-43毒性。库进行转化并接种到葡萄糖和半乳糖的板（ 图2），以评估在屏幕的严格性。单菌落，选择并且该菌株，使用5-FOA消除的Hsp104质粒计数器选中。将这些菌株再评估以确认毒性是由于TDP-43的单独，没有的Hsp104变体。在初始屏幕中选择的变体的一个子集的点样分析表明，所选择的4菌落，2显示的Hsp104介导的TDP-43的毒性抑制，1呈假阳性和1个显示毒性增强以下5-FOA的处理（ 图3）。 2真实命中然后，通过菌落PCR测序以鉴定中间结构域的突变（ 图4）。一旦这些突变进行鉴定，所述的Hsp104突变构建<html>重新在亲代质粒的Hsp104通过位点定向诱变，以确认毒性的抑制。使用这些方法的选择变体后来被证实为抑制聚集在酵母中，在生化分析清楚预制聚集，并抑制了C.多巴胺能神经退行性疾病PD 17的线虫模型。 <img alt="图1" fo:content-width="7in" src="/files/ftp_upload/52089/52089fig1highres.jpg" width="700" /> 图1:流的图表用于分离增效的Hsp104变体的Hsp104库（URA3标记，GAL1启动子）转化在含病基板（HIS3标记，GAL1启动子），酵母和通过电镀在诱导培养基筛选毒性抑制剂。潜在的命中然后再次使用5-FOA的反选择步骤，以消除非特异性毒性抑制剂筛选。在这第二步骤中选择的变体，然后通过菌落PCR测序。<html> HREF ="htt​​ps://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52089/52089fig1large.jpg"TARGET ="_空白"&gt;请点击这里查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52089/52089fig2highres.jpg" /> 图2:筛选毒性抑制酵母菌体与pAG303GAL-TDP-43和pAG416GAL-的Hsp104文库接种到葡萄糖（抑制）或半乳糖（诱导）的介质。 TDP-43是剧毒，所以很少的Hsp104变体能够抑制该毒性的，并且该屏幕是非常严格的。单个菌落被选择作为从半乳糖板对5-FOA的二次筛选命中。大，小菌落通常获得的，​​但我们并没有观察到，决定如何菌落大小与活性的变化趋势。 s.jpg"/&gt; 图3:5-FOA的二次筛选酵母治疗与5-FOA的打击选择的质粒的Hsp104点样测定法进行评估。株生长在SRaff，他的媒体，连续稀释5倍，并发现一式两份到SD-他和SGal，他的板。 HSP104 A503V是我们以前已经验证，并在这里展示，并附有单独的17矢量控制一个真正的打击。行3和4是保留的Hsp104抑制器的TDP-43的毒性下列损失文库命中。第5行示出了假阳性，在那里，TDP-43不再是有毒的，因此，非特异性毒性抑制剂的存在。行图6示出的菌株即是更具毒性比TDP-43通常是，可能是由于5-FOA的毒性。该菌株仍然可以被测序，但可能不是一个有效命中。 <img alt="图4" fo:content-width="3in" src="/files/ftp_upload/52089/52089fig4highres.jpg" width="300" /> 图4.肛门yzing测序结果。选择的酵母通常包含多个质粒。因此，以下的菌落PCR产物测序，必须小心，以确保任何突变都没有在色谱忽视。在这个色谱图，两个潜在的突变（记为*）可能会被忽略。在第一个实例，有A和T以及在所述第二混合物，T和C的混合物

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

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.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Universidad San Sebastian

Research

JoVE Journal

Biology

8.1K 次观看.  Perelman School of Medicine at the University of Pennsylvania. 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.

视频: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

科研

癌症研究

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

遗传学

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫与感染

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

生物学

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估:兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

隔离增效变种的Hsp104利用酵母Proteinopathy模型

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