Name: assays for the degradation of misfolded proteins in cells
Uploaded: 2016-08-28T15:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about Assays for the Degradation of Misfolded Proteins in Cells at JoVE.com

We thank S. Raychaudhuri for providing the destabilized firefly luciferase mutant plasmid, and A. Glavis-bloom and N. Charan for technical assistance. This work was supported, in part, by grants from NIH (CA088868, GM060911, and CA182675).

1.试剂的制备<ol><li>制备细胞裂解缓冲液（50mM的Tris，pH值8.8，100mM NaCl的，5mM的MgCl 2的，0.5％的NP-40）。补充2毫摩尔DTT，1×完全蛋白酶的鸡尾酒，和250国际单位/毫升BENZONASE使用前。 </li><li>制备粒料缓冲液（20mM的Tris，pH值8.0，15mM的MgCl 2的）。补充2毫摩尔DTT，使用前1个完整的蛋白酶的鸡尾酒和250国际单位/毫升BENZONASE。 </li><li>制备3倍沸腾缓冲液（6％SDS，20毫摩尔Tris，pH8.0）中。使用前补充150毫摩尔DTT。 </li><li>准备使用荧光酶标仪检测读卡器低荧光DMEM培养基。混合的25mM葡萄糖，0.4mM的甘氨酸，0.4毫精氨酸，0.2mM的半胱氨酸，4.0 2mM谷氨酰胺，0.2mM的组氨酸，0.8毫异亮氨酸，0.8毫亮氨酸，0.8毫赖氨酸，0.2mM的甲硫氨酸，0.4mM的苯丙氨酸，0.4mM的丝氨酸，0.8毫苏氨酸，0.078毫色氨酸，0.4mM的酪氨酸，0.8毫缬氨酸，1.8毫氯化钙 ，0.81毫硫酸镁 ，5.33毫米氯化钾，44.0毫碳酸氢钠 ，110 mM氯化钠，0.9毫加入NaH 2 PO 4。调整的使用盐酸或NaOH至pH 7.4溶液的pH值。消毒通过过滤介质。 注意:此培养基含有除了铁具有高葡萄糖的标准DMEM培养基组分（NO 3）3，维生素和酚红被省略。酚红，核黄素和吡哆醛在定期的DMEM培养基与检测荧光信号21的显著干扰。如果没有这些成分的培养基是成功的活细胞成像GFP至关重要。当冷藏保存介质是12个月是稳定的。 </li></ol> Atxn1 82Q GFP的2降解测定<ol><li>板大约3×10 5个 HeLa细胞与DMEM介质35 MM平板补充有10％胎牛血清（FBS），使得后O / N培养细胞生长至40-60％汇合在转染时。 注意:需要板的数量是基于时间点和治疗D中的数下面旁切。 </li><li>转染的HeLa细胞与Atxn1 82Q-GFP / pRK5质粒0.3微克到根据制造商的指令9每孔用转染试剂。使含有DNA和转染试剂主染混合物和分装它每口井。 </li><li> 4-5小时后转染，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。返回细胞回孵化后成像。 注意:此时请遵守在核两种扩散GFP信号和GFP信号的小斑点。 </li><li>通过真空抽吸除去培养基，加入2毫升的新鲜的DMEM含有50微克/毫升CHX。处理细胞用于与收获前CHX 0，4，8，第12和16小时。为了检验蛋白酶体降解，包括在16小时作为对照处理的一个板蛋白酶体抑制剂MG132（10μM）。 </li><li>在每个时间点收获细胞的一个极板。通过负压吸引的移除介质ð用3毫升冰冷的1X磷酸盐缓冲盐水（PBS）洗涤板两次。管理单元冻结干冰板。 </li><li>最后时间点（16小时）后，刮去冷冻细胞（来自所有时间点）到150微升冰冷的细胞裂解缓冲液，并在冰上温育30分钟。 </li><li>离心在台式离心机的细胞裂解物在17000×g离心在4℃下15分钟。 </li><li>将上清转移，其中包含NP-40的可溶性（NS）蛋白，使用移液管的另一个管。 注:使用为上清液如果需要通过Bradford法测定蛋白质的浓度。 </li><li>轻轻加入约200μl的1×PBS中的管而不会干扰粒料冲洗粒料。小心地负压吸引或吸管除去PBS。重悬它们在150微升冰冷的细胞沉淀缓冲液中，接着15-30分钟温育在冰上。 注:重悬沉淀馏分含有NP-40的不溶物（NI）的蛋白质。参见图1B的诊断洗涤剂分馏的RAM。 </li><li>添加75微升3倍沸腾缓冲器的成NS个级分和NP-40的不溶物（NI）的级分从粒料再悬浮。加热该样品在95℃，5分钟的加热块上。 注:竹丛中的NP-40不溶性馏分加热和样品将明朗化后解散。 </li><li>添加SDS-凝胶上样缓冲液至煮沸的NS和NI的等分试样。加载来自所有时间点到SDS-PAGE凝胶收集的样品的等体积。从样品的体积装载对应于大约20微克的NS中的时间0。注收集:NS，以及SDS可溶性（SS）蛋白来自NI派，可以通过SDS分离胶来解决。与此相反，来自NI馏分SDS抗性（SR）聚集体卡在凝胶上样孔中（ 图1B）。为了提高免疫印迹检测，NI的双卷可装载。 </li><li>通过使用抗GFP抗体和增强化学发光（ECL）免疫印迹检测NS和SS Atxn1 82Q-GFP。 注意:使用此协议时，Atxn1 82Q在SS分数一般不太相比NS分数。是为了得到理想的信号再ECL曝光。 </li><li>从通过使用斑点印迹装置（ 图1B）滤波器相位差测定沉淀馏分审查SR Atxn1 82Q。简单地说，设立了点印迹装置拿着0.2微米醋酸纤维素膜。负载80-120微升煮沸的NI的入点印迹装置的各孔中。 注:由于在细胞中仅形成的SR聚集体的少量的，为对斑点印迹测定中，加载的SR分数是用于Western印迹分析的NS馏分的体积的约10-15倍的体积。 <ol><li>通过真空膜过滤样品后，停留在膜Atxn1 82Q-GFP聚集体可通过抗GFP免疫印迹9,12,22来检测。 注:请参阅之前的报道9,12,22过滤器阻滞实验的详细描述。此步骤是可选因为相对于按照本协议描述的短期转SS和NS分数SR Atxn1 82Q是最小的。此外，SR Atxn1 82Q的电平在很大程度上仍超过CHX追逐（ 图2B）相同。然而，它仍然是至关重要的，审查的SS Atxn1 82Q的量是否会受到影响随着时间的推移对在最初的实验中任何药物治疗或基因表达。 SS蛋白物种留在SDS-PAGE凝胶上样孔中可以通过免疫印迹进行检测。然而，该方法是不太敏感，并产生更多不同的结果（ 图1B）。 </li></ol></li></ol> NLS-荧光素酶-GFP的使用SDS-PAGE和Western Blot 3.降解测定<ol><li>板大约3×10 5个 HeLa细胞与DMEM介质35 MM平板补充有10％胎牛血清（FBS）。 O / N培养后，在转染时达到40-60％的汇合。所需板的数量是基于叔数IME点和治疗如下所述。 </li><li>转染的HeLa细胞用1.0微克NLS萤光素酶-GFP / pRK5质粒导入根据制造商的指令9每孔用转染试剂。使包含每个等分DNA和转染试剂以及一个主染混合物。 </li><li> O / N转（小时约15）后，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。返回细胞回孵化后成像。 注:扩散核GFP信号所用的大部分细胞（70％）的被观察到。细胞的一小部分（5％）有核聚集。 </li><li>通过真空抽吸除去培养基，加入2毫升的新鲜的DMEM含有50微克/毫升CHX。处理细胞与收获前CHX 0，1.5,3，4.5和6小时。为了检验蛋白酶体降解，包括在6小时作为对照处理的一个板附加蛋白酶体抑制剂MG132（10μM）。 </li><li>在每个时间点收获细胞的一个极板。除去由真空抽吸培养基并用3ml冰冷的磷酸盐缓冲盐水（PBS）洗涤板两次。管理单元冻结干冰板。 </li><li>最后时间点（6小时）结束后，细胞冷冻于所有时间点都刮入150微升冰冷的细胞裂解缓冲液，并在冰上温育30分钟。 </li><li>加SDS凝胶加样缓冲液以全细胞裂解物的2％的SDS的终浓度和50mM DTT。孵育一个热块上的样品在95℃下5分钟。 </li><li>通过SDS-PAGE和使用抗GFP抗体免疫印迹分析的NLS萤光素酶-GFP。加载来自所有时间点到SDS-PAGE凝胶收集的样品的等体积。卷装相当于大约20微克从样本在时间0收集全细胞裂解物的。 </li></ol>使用荧光酶标仪NLS-荧光素酶-GFP 4.实时降解测定<ol><li>种子约1×10 4个HeLa细胞与透明底的黑色96-孔组织培养板中。 O / N培养后，在转染时达到50-70％汇合。 注:60微升含有完全悬浮HeLa细胞培养基的直接接种到各孔中。不要播种后添加额外的媒体或岩石板上来回，否则电池可能会分布不均。 </li><li>转染的HeLa细胞与NLS-萤光素酶-GFP / pRK5质粒9的0.05-0.1微克到根据制造商的指示向各孔使用转染试剂。使含有DNA和转染试剂主染混合物和分装它每口井。三个井用细胞不与DNA，作为为每个处理条件背景荧光信号控制转染。 注:另一种方式，以减少染变化是播种之前转染细胞。然而，转染错折叠蛋白的细胞的大部分小号不重视或使用此方法显示减少生存能力。它很可能是一些细胞可能经不起表达毒性错误折叠的蛋白质时所造成的胰蛋白酶消化的应力。其结果是，总的荧光信号显著降低。 </li><li>转染后20-24小时，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。 </li><li>删除由负压吸引的网上平台。添加约200μl的1×PBS中，以每个孔，然后吸出它来除去DMEM培养基的剩余量。 </li><li>添加60微升低荧光DMEM培养基有5％FBS和50μg/ ml的放线菌酮。为了检验蛋白酶体降解，包括在一组样品额外蛋白酶体抑制剂MG132（10μM）。集井的一式三份的每个处理/条件。 注:MG132治疗被包括作为蛋白酶体降解的控制，因为它是非常重要的排除可能会导致FLUO的压降等因素rescence信号，包括细胞死亡和荧光淬灭。 </li><li>立即测量加入CHX后，荧光酶标仪GFP的荧光信号。 注:软件测量设置如表1所示。 </li><li>读板每隔一小时可达8-10小时。看完后，退回板回细胞培养孵化器。 </li><li>将数据导出为电子表格文件。使用平均值为每个多读取以及荧光强度。正常化每个数据点的值，以时间0的在各组的平均值。积归一化的荧光强度随着时间的推移， 如图4A和4B，以及图5，右面板。 </li><li>执行使用两因素ANOVA重复测量23两个条件之间降解率的统计分析。 注:在这种分析中，"荧光强度"是，而"treatm因变量耳鼻喉科条件"和"时间"是两个因素，而是在单独的时间点进行比较的数据，双向ANOVA重复测量分析在整个时间过程的两个治疗组之间的差异。 </li></ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+degradation+of+truncated+polyglutamine+proteins+by+the+proteasome.">Inefficient degradation of truncated polyglutamine proteins by the proteasome.</a> EMBO J. 23, 4307-4318 (2004).</li><li>Verhoef, L. G., Lindsten, K., Masucci, M. G., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregate+formation+inhibits+proteasomal+degradation+of+polyglutamine+proteins.">Aggregate formation inhibits proteasomal degradation of polyglutamine proteins.</a> Human molecular genetics. 11, 2689-2700 (2002).</li><li>Jana, N. R., Zemskov, E. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibitor+PS-341+induces+apoptosis+through+induction+of+endoplasmic+reticulum+stress-reactive+oxygen+species+in+head+and+neck+squamous+cell+carcinoma+cells.">Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells.</a> Molecular and cellular biology. 24, 9695-9704 (2004).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasarcoplasmic+amyloidosis+impairs+proteolytic+function+of+proteasomes+in+cardiomyocytes+by+compromising+substrate+uptake.">Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake.</a> Circulation research. 97, 1018-1026 (2005).</li><li>Link, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+green+fluorescent+protein+into+a+toxic,+aggregation-prone+protein+by+C-terminal+addition+of+a+short+peptide.">Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.</a> The Journal of biological chemistry. 281, 1808-1816 (2006).</li></ol>

The authors declare that they have no competing financial interests.

调节错误折叠的蛋白质的降解机制是用于维持细胞蛋白质的稳态必不可少的，并且它们可能代表用于治疗神经变性疾病和其他蛋白质错误折叠的疾病有价值的药物靶标。这里，检查错误折叠的蛋白质的降解测定法所述，用致病Atxn1蛋白（Atxn1 82Q）和核局部化的萤光素酶突变体（NLS-LucDM）作为例子。 为了检查降解Atxn1 82Q，具有半衰期超过18小时，我们介绍了细胞分离与经典CHX <su...

蛋白是在细胞中最丰富的大分子，和它们在几乎所有的生物过程中起重要作用。大多数蛋白的生物活性，需要其折叠成和维护，天然的三维结构。与异常的构象的蛋白不仅失去其正常功能，而且还常常形成削弱其它蛋白质的功能和对细胞有毒性，1,2-可溶性寡聚物种或聚集体。为了抵消蛋白质错误折叠，细胞同时采用分子伴侣，这有助于展开或部分折叠多肽达到自己的天然构象，和降解途径，从而消除错误折叠的蛋白质3。给出的复杂性和折叠处理的随机性质，蛋白错折叠是不可避免的，而且它可能无法在突变，生物合成的错误，以及翻译后的损害1的情况是相反的。因此，细胞最终依靠degradat离子通道以维持其蛋白质的质量。 细胞蛋白质的质量控制（PQC）系统的重要性，通过蛋白质错误折叠的疾病，包括癌症，糖尿病，和许多神经退行性疾病如阿尔茨海默氏病，帕金森氏病，肌萎缩性侧索硬化症，亨廷顿氏病（HD）的患病率强调，和小脑萎缩症（SCA）4,5。例如，在肿瘤抑制基因p53的突变是唯一最频繁的遗传病变肿瘤，具有〜所有病例6 50-70％相关联。 p53基因突变的相当大的部分是改变p53的构象的错义突变，导致聚集体7的形成。此外，扩大的polyQ绵延蛋白基因和病理与HD和SCA相关。当的polyQ伸展在受影响的蛋白质的长度超过一定THRES这些进步和常常是致命的疾病表现持有，并作为的polyQ拉伸的长度延长8变得越来越严重。 用于治疗这些疾病的有吸引力的方法是将治疗加强蜂窝PQC系统，特别是降解途径。然而，涉及有缺陷的蛋白质，特别是那些在哺乳动物细胞中的降解途径，仍然知之甚少。尽管已经认识到，蛋白酶体为错误折叠的蛋白质的降解非常重要，一个至关重要的问题仍然是未定义:蛋白质如何错误折叠被特异性识别和针对降解。此外，尽管PQC系统在细胞区室，包括细胞质中，内质网和线粒体已经确定，在细胞核中的PQC系统仍不清楚2。 由我们实验室最近的研究已经确定了在细胞核中识别并降低各种错折叠蛋白的一个系统哺乳动物细胞中9。这个系统由早幼粒细胞蛋白（PML），核蛋白质和三方含基序（TRIM）蛋白质家族的一员，并RNF4，含蛋白质的RING结构域的。 PML等几个TRIM蛋白，具有SUMO（小泛素样修饰符）E3连接酶的活性，有利于蛋白SUMO化10的特异性和效率。 RNF4属于一个小团体SUMO-针对性的泛素连接酶（STUbL），其中包含在除了能提供他们泛素连接酶的活性在11 RING结构域的一个或多个相扑相互作用基序（SIMS）的。我们发现，PML特异性识别通过，可以在错误折叠的蛋白质辨别特色鲜明离散底物识别位点错误折叠的蛋白质。结合后，PML标签错误折叠与SUMO2 / 3的聚链，两个几乎相同的哺乳动物SUMO蛋白可以形成由于内部SUMO化位点的存在聚链蛋白质。小号UMOylated错误折叠的蛋白质，然后通过RNF4，从而导致它们的泛素化和蛋白酶体降解的认可。我们进一步证明了PML-RNF4系统是针对神经变性保护重要的，因为缺乏在PML加剧SCA类型的小鼠模型1（SCA1）9的行为和神经病理学缺陷。 若要从可调节蛋白水平等细胞机制区分蛋白质降解，蛋白质周转率测量9。其中最常用的方法，以确定蛋白质周转是脉冲追踪和放线菌酮（CHX）追。这两种方法考察随着时间的推移，分别翻译，精通细胞和翻译，抑制细胞的总利息预先存在的蛋白质感兴趣的放射性同位素标记的蛋白质。但是，对于学习致病和错误折叠倾向的蛋白质的一个主要的挑战是，这些蛋白质的半衰期可以是非常罗NG。例如，共济失调蛋白1，共济失调蛋白7，亨廷顿，α突触核蛋白，和TDP-43都具有超过12的半衰期至24小时9,12-16。这些蛋白质的慢流动率排除使用CHX追分析的，因为携带这些蛋白质的细胞可能无法生存延长翻译抑制，特别是因为错误折叠的蛋白质本身可以是细胞毒性很强。用同位素标记的脉冲追踪分析还可以是对于那些高度聚集倾向的蛋白质具有挑战性。最脉冲追踪测定依赖于免疫沉淀到感兴趣的蛋白质从也放射性标记的所有其他蛋白质分离。此过程通常包括冗长免疫沉淀和洗涤过程，在此期间，SDS不溶聚集体可以形成，使得用SDS-PAGE电泳不精确的分析。 这里用一个缓慢的周转率说明9协议分析核错误折叠的蛋白质。包含的82个谷氨酰胺（Atxn1 82Q）拉伸共济失调蛋白-1（Atxn1）的致病形式用于此目的8。当在细胞中表达的Atxn1 82Q形式显微镜可见夹杂物的增强型绿色荧光蛋白（GFP）融合在细胞核中（ 图1A）。脉冲追踪分析表明Ataxn1 82Q的半衰期为18小时以上，9。 Atxn1 82Q是由具有不同特性的错折叠的构象，以及具有天然构象物种物种。它很可能是这些物种以不同的速率退化，并因此应该被单独分析。裂解物从Atxn1 82Q-GFP表达细胞被分馏成NP-40的水溶性（可溶于或生理盐水，可能代表天然蛋白质或错误折叠的单体/低聚物的蛋白）和NP-40的不溶性（NI，凝集/错折叠）部分。后者可进一步分为SDS可溶性（SS;可能无序集合体）或SDS抗性（SR;可能淀粉样蛋白原纤维）馏分（ 图1B）。 NS和SS馏分可以通过SDS-PAGE，随后通过Western印迹进行分析，而对SR分数可以通过过滤相位差测定，随后通过免疫印迹进行检测。 CHX追与洗涤剂分馏方法相结合，并且发现SS Atxn1 82Q的半衰期比NS Atxn1 82Q和总Atxn1 82Q（ 图2A）的短得多，这表明在SS馏分可以容易地识别和降解在细胞中9。因此，该方法提供了一个有力的工具来研究错误折叠的蛋白质的动态，并比较它们的降解模式。 我们还描述了适合于用于识别大分子或小分子化合物可以调节错误折叠的蛋白质的降解的高通量筛选的方法。此方法是基于萤火虫荧光素酶（LucDM）17，一个模型伴侣衬底的构象失稳突变体。我们有保险丝ðLucDM到一个核定位信号（NLS）来探测在此细胞区室的PQC系统，和GFP（NLS-LucDM-GFP）进行检测的便利性。 NLS-LucDM-GFP形式显微镜中的细胞（ 图3A）的一小部分可见核聚集体。类似Atxn1 82Q，NLS-LucDM-GFP-而不是它的野生型对应物NLS-LucWT-GFP-由SUMO2 / 3改性和调节由PML-RNF4通路9。 NLS-LucDM-GFP也形成SS和SR物种，虽然SS和SR物种的相对量相比NS是最小的，使用下文协议3所述的条件。为了简化分析，我们只分析SDS可溶性LucDM（包括NS和SS分数两者）通过SDS-PAGE和免疫印迹。重要的是，CHX追检测显示SDS可溶性NLS-LucDM-GFP的半衰期比其野生型对应物（ 图3B）的短得多，这表明LucDM为承认和降解系统中的特定基板错误折叠的蛋白质。 LucDM-GFP的降解导致整体荧光信号的显著下跌。因此，我们还开发了使用基于荧光微量测定细胞LucDM-GFP的实时检测的协议。许多高通量筛选（HTS）系统的药物或基因修饰的细胞聚集和细胞活力造成的异常蛋白质18-20发展。然而，很少HTSS是专门用于在哺乳动物细胞靶向降解而设计的。此协议可作为蛋白质表达，击倒，和药物治疗对细胞错折叠蛋白质降解的影响快速和大规模分析一个健壮的系统。使用HeLa细胞作为例子，下面我们描述了协议，这两个错误折叠的蛋白质的分析。该测定法还可以应用到其他的细胞系，虽然转染的条件和时间过程中可能需要对个别细胞系进行优化。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Life Technologies</td><td>11995-092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668019</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>NP40S-500ML</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>MG132</td><td>Sigma-Aldrich</td><td>M8699</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma-Aldrich</td><td>C7698</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Scientific</td><td>45000232</td><td></td></tr><tr><td>Complete Protease Inhibitor Cocktail Tablets</td><td>Roche Boehringer Mannheim</td><td>4693159001</td><td></td></tr><tr><td>Bio-Dot Apparatus&amp;nbsp;</td><td>Bio-Rad</td><td>1706545</td><td></td></tr><tr><td>Living Colors GFP Monoclonal Antibody</td><td>Clonetech</td><td>632375</td><td></td></tr><tr><td>Anti-Actin mAb Rabbit, IgG</td><td>Sigma-Aldrich</td><td>A45060-200UL</td><td></td></tr><tr><td>Amino acids</td><td>Sigma-Aldrich</td><td></td><td>Amino acids are used for making low fluorecence culturing medium</td></tr><tr><td>Olympus IX-81&amp;nbsp; Inverted Fluorescence Microscope</td><td>Olympus</td><td>IX71/IX81</td><td></td></tr><tr><td>96 Well Black TC Plate w/ Transluscent Clear Bottom</td><td>Sigma-Greiner</td><td>89135-048</td><td></td></tr><tr><td>Fluorescence Bottom Plate Reader Infinite 200&amp;reg; PRO</td><td>TECAN</td><td>Infinite 200&amp;reg; PRO</td><td></td></tr><tr><td>Cellulose acetate membrane 0.2 &amp;micro;m</td><td>Sterlitech</td><td>CA023001</td><td></td></tr><tr><td>Prism 5</td><td>GraphPad</td><td></td><td>Statistical analysis software</td></tr></tbody></table>

assays for the degradation of misfolded proteins in cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

在稳定状态下的分析，显微镜可见Atxn1 82Q-GFP核聚集体可以在30观察到- HeLa细胞的50％的转染（ 图1A）后20小时。使用抗GFP抗体的NS和SS级分的Western印迹分析显示100 kDa和150 kDa的标记之间Atxn1 82Q-GFP的一个独特的带，对应于该蛋白质的分子量（ 图1B）。 Atxn1 82Q-GFP在SR分数可以通过过滤相位差测定法，或通过SDS-PAGE和免疫印迹如在浓缩胶（ 图1B）</st...

Watch this Scientific Journal Video about Assays for the Degradation of Misfolded Proteins in Cells at JoVE.com

Assays for the Degradation of Misfolded Proteins in Cells

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

assays-for-the-degradation-of-misfolded-proteins-in-cells

Research

JoVE Journal

Biology

11.9K 次观看.  University of Pennsylvania Perelman School of Medicine. This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening. 

视频: Assays for the Degradation of Misfolded Proteins in Cells

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

科研

生物化学

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

神经科学

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

生物学

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. <a href="https://www.jove.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

蛋白质聚集体形成测定:一种在蛋白酶体抑制剂诱导下检测和定量培养细胞中蛋白质聚集的方法

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

Source: Guo, L. et al., <a href="https://app.jove.com/v/54266">Assays for the Degradation of Misfolded Proteins in Cells.</a> J. Vis. Exp. (2016).
This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.

易错折叠的蛋白质降解测定:一种使用放线菌酮处理和去污剂分级分离监测错误折叠蛋白质降解的技术

Source: Guo, L. et al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016).
This video describes a degradation assay for ...

测定法错误折叠的蛋白质在细胞中的降解

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可溶性和不溶性杭丁顿蛋白的分离分解

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

在活细胞中蛋白质聚集的4D成像

耦合检测监控蛋白复性

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

测定蛋白酶体降解的植物无细胞系统

记者的增长分析的蛋白质降解的系统分析

成长型测定和遗传需求生化确认的蛋白质降解

蛋白质聚集体形成测定:一种在蛋白酶体抑制剂诱导下检测和定量培养细胞中蛋白质聚集的方法

易错折叠的蛋白质降解测定:一种使用放线菌酮处理和去污剂分级分离监测错误折叠蛋白质降解的技术