Name: reporter-based growth assay for systematic analysis of protein degradation
Uploaded: 2014-11-06T13:00:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

We thank Dr. Yuval Reiss and Dr. William Breuer for critically reviewing the manuscript and Omri Alfassy for helping in the development of the Ura3-GFP screen assay. We also thank Dr. M. Hochstrasser and Dr. R. Kulka for plasmids and strains. This work was funded by the Israeli Academy of Sciences (grant 786/08) and by the United States-Israel Binational Scientific foundation (grant 2011253).

1.细胞培养<ol><li>变换相应的营养缺陷型的酵母细胞，诸如Try467（ 表2），用含有（a）一种酵母URA3 -degron融合和（b）额外的代谢标记物的质粒的选择和维护的质粒。 注:合适的质粒的实例是YDpK-MET25p-Deg1-FLAG-Vma12-的Ura3（LYS2）（Deg1-紫外线）这是一个含有一种融合蛋白，其包含Deg1的酵母整合质粒的-从酵母转录得到的降解决定子。因子MAT2 17，FLAG表位-为检测通过免疫印迹法，Vma12 -稳定的ER蛋白，和的Ura3 15,18（ 图3A）。 （请参阅表3本研究和对合适的质粒的额外实施例中使用的质粒）。 </li><li>对于质粒的选择和维护，保持细胞在琼脂平板在合适的合成定义（SD）中缺少的氨基酸选择标记<s向上&gt; 19（例如，Deg1 -UV被选择并下的SD-LYS选择性培养基维持）。 </li><li>在对质粒保持一个合适的选择性的SD培养基上生长的细胞O / N在30℃下，直至稳定期为止。生长的细胞在试管中或在96孔板中，根据样本的数目要被检查（第2部分）。 </li></ol> 2.细胞制备方法分析<ol><li>当少量样品（N≤15）是待测定，稀释细胞并更换培养基如下: <ol><li>稀释50μl的细胞从一种O / N培养物用150微升每孔的SD培养基中，在96孔板（透明底）。注意，第一个样品被指定只作为空白包含介质。 </li><li>测定用酶标仪在96孔板中的样品的OD 600值。 </li><li>计算实际的OD 600在各孔中通过乘以OD 600读数乘以稀释因子（4个），从的whICH空白读数的值中减去。由0.55分，得到为1cm根据比尔 - 朗伯定律计算出的路径长度的归一化所得的值。注意，测量OD 600 200微升的细胞在标准的96孔板，当这个值是恒定的。 </li><li>计算出所需的用于获得的酵母细胞在量相当于OD 600为0.25，并将其转移到Eppendorf管中的准确体积。 </li><li>降速细胞以高速（12,000 xg离心），持续1分钟。 </li><li>弃上清，重悬细胞于1ml适当的选择性培养基（见3.1节），以获得OD 600为0.25。 </li><li>转移200微升稀释的菌株，以一个新的井在96孔板中。 </li></ol></li><li>当大量的样本（N&gt; 15）进行测试，稀释细胞并替换无需事先的OD 600测量的介质，具体如下: <ol><li>转让10微升静止细胞生长的O / N在96孔板到一个新的96孔板中含有合适的选择性培养基的190微升（稀释1:20）（见3.1节）。 </li></ol></li></ol>液体培养在选择性条件3.生长动力学（GILS） <ol><li>使用下面的媒体使用的Ura3，降解决定子记者成长的测量: <ol><li>对于非限定性条件下酵母菌的生长，利用质粒选择性SD存储介质的阳性对照（参见1.2节）。如果没有质粒的选择是必需的，例如，使用整合质粒如Deg1 -UV，含有所有必需氨基酸（SD完整）的SD培养基中时，可以使用。 </li><li>对于实验样品的测量限制条件下生长，使用SD-URA中。 </li></ol></li><li>设置一个多模酶标仪到:30℃，外径600测量的15分钟间隔的温育温度，1分钟每7分钟的轨道和线性振荡周期&lt;BR /&gt;注:此双模摇动优化，以获得细胞的均匀分布;然而，其他的晃动模式，甚至没有晃动可以正常工作。 </li><li>在孵育的细胞中，在30℃的微量板读数器的O / N或直到细胞达到稳定期（12-24小时）。 </li><li>原始数据导出到电子表格文件。 </li><li>确定细胞加倍的人口规模使用MDTcalc，即计算最小时间（小时）定制的软件程序，酵母生长动力学（最小的倍增时间（MDT）;有关详细信息，请参见第4节）。 </li></ol>最小的倍增时间4计算（MDT）:在MDTcalc算法的原理注:该MDTcalc算法的原理，使用在SD完全培养基中生长Try467酵母细胞（ 表2）的有代表性的样品，示于表1和图1中 ，示出了数据的电子表格和生长的地块，respectively。 <ol><li>减去的OD 600为空白值，在相应的孔获得，从每个样品中获得良好的读数。由0.55分所得的差异（对于96孔板），以校正所述光路长度。绘制的最后的值（ 表1中 ，反式。）对时间，以产生实际的酵母的生长曲线（OD 600 /小时）（ 图1A）。 </li><li>计算日志2（OD 600）为每个经变换的值以对数生长期的转化为线性曲线（ 图1B）。 </li><li>计算10的时间点的时间间隔（N = 10）开始于时刻0和移动一个时间点的时间，直到Ñ&lt;10的斜率（ 表1，斜率; 图1C）。 </li><li>计算每个斜率的倒数的值，以获得加倍所需的细胞群的时间（ 表1中 ，1 /斜率; 图1C）。计算最小1 /斜率值，以确定MDT的（ 表1，黑色矩形; 图1C）。提取所计算的MDT为从选定的时间间隔内的细胞样品中（ 表1中，1 /斜率，黑色矩形）（ 表1中，由红色矩形概述， 图1B，图1D）。 注:在SD-URA培养基中的细胞生长速率成反比的MDT。 </li></ol> 5.使用MDTcalc <ol><li>补充的程序的文件复制到在计算机中的指定文件夹。 </li><li>确保保存包含增长数据的电子表格文件，并关闭。打开MDTcalc应用程序，看看屏幕出现的开始。 <ol><li>插入所需信息，这表现在图2中: <ol><li>在"文件路径"，单击矩形上找到电子表格文件的权利。 </li><li>在"表名称"，其中t编写电子表格名称他的原始数据所在的位置。 </li><li>下"起始位置"中，插入的第一样本的时间0的电子表格坐标。 </li><li>下"时间列&#39;，插入信限定时间点列的位置（时间用秒来提供）。 </li><li>根据&#39;空白列&#39;，插入字母定义空白栏的位置（通常是但不一定是在A1孔在96孔板中）。 </li><li>在"OD值"，选择所需的MDT计算的开始（通常0.15-0.25）的最低转化OD 600值。设置此值可防止MDT的计算由于过度稀释培养的滞后期，这样MDT只为达到规定的OD 600值以上的样本计算的。 </li></ol></li><li>一旦最后的值被输入，按下屏幕底部的"计算"按钮。确保在正确的进度条逐渐变化到克颖。在计算过程中不要打开电子表格文件。 </li><li>导出的两组数据，可自动显示在屏幕上，在完成了的MDT计算到电子表格。确保该数据包括:（a）在各孔中传递的最小OD值测试（如在节5.2.1.6中设置）和（b）从所述的MDT来计算的时间间隔的起始时间点的MDT值（开始时间）。 </li></ol></li></ol>

<ol>
	<li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annual review of biochemistry. 67, 425-479 (1998).</li><li>Ravid, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+degradation+signals+in+the+ubiquitin-proteasome+system.">Diversity of degradation signals in the ubiquitin-proteasome system.</a> Nature reviews. Molecular cell biology. 9, 679-689 (2008).</li><li>Willems, A. R., 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=SCF+ubiquitin+protein+ligases+and+phosphorylation-dependent+proteolysis.">SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.</a> Philos Trans R Soc Lond B Biol Sci. 354, 1533-1550 (1999).</li><li>Tyers, M., Jorgensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+and+the+cell+cycle:+with+this+RING+I+do+thee+destroy.">Proteolysis and the cell cycle: with this RING I do thee destroy.</a> Curr Opin Genet Dev. 10, 54-64 (2000).</li><li>Johnson, P. R., Swanson, R., Rakhilina, L., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signal+masking+by+heterodimerization+of+MAT&#945;2+and+MAT+a1+blocks+their+mutual+destruction+by+the+ubiquitin-proteasome+pathway.">Degradation signal masking by heterodimerization of MAT&#945;2 and MAT a1 blocks their mutual destruction by the ubiquitin-proteasome pathway.</a> Cell. 94, 217-227 (1998).</li><li>Furth, N., 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=Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum/nuclear+envelope.">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Molecular Biology of the Cell. 22, 4726-4739 (2011).</li><li>Fredrickson, E. K., Gallagher, P. S., Clowes Candadai, S. V., Gardner, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+recognition+in+nuclear+protein+quality+control+degradation+is+governed+by+exposed+hydrophobicity+that+correlates+with+aggregation+and+insolubility.">Substrate recognition in nuclear protein quality control degradation is governed by exposed hydrophobicity that correlates with aggregation and insolubility.</a> J Biol Chem. 10, (2013).</li><li>Rosenbaum, J. C., 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=Disorder+Targets+Misorder+in+Nuclear+Quality+Control+A+Disordered+Ubiquitin+Ligase+Directly+Recognizes+Its+Misfolded+Substrates.">Disorder Targets Misorder in Nuclear Quality Control A Disordered Ubiquitin Ligase Directly Recognizes Its Misfolded Substrates.</a> Molecular cell. 41, 93-106 (2011).</li><li>Ciechanover, A., Hod, Y., Hershko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heat-stable+polypeptide+component+of+an+ATP-dependent+proteolytic+system+from+reticulocytes.">A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.</a> Biochemica., &amp; Biophysical Research Communications. 81, 1100-1105 (1978).</li><li>Finley, D., Ulrich, H. D., Sommer, T., Kaiser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+system+of+Saccharomyces+cerevisiae.">The ubiquitin-proteasome system of Saccharomyces cerevisiae.</a> Genetics. 192, 319-360 (2012).</li><li>Klis, F. M., Boorsma, A., De Groot, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+construction+in+Saccharomyces+cerevisiae.">Cell wall construction in Saccharomyces cerevisiae.</a> Yeast. 23, 185-202 (2006).</li><li>Zhou, W., Ryan, J. J., Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analyses+of+sumoylated+proteins+in+Saccharomyces+cerevisiae.+Induction+of+protein+sumoylation+by+cellular+stresses.">Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.</a> J Biol Chem. 279, 32262-32268 (2004).</li><li>Alani, E., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+type+of+fusion+analysis+applicable+to+many+organisms:+protein+fusions+to+the+URA3+gene+of+yeast.">A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.</a> Genetics. 117, 5-12 (1987).</li><li>Swanson, R., Locher, M., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+ubiquitin+ligase+of+the+nuclear+envelope/endoplasmic+reticulum+that+functions+in+both+ER-associated+and+Matalpha2+repressor+degradation.">A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.</a> Genes Dev. 15, 2660-2674 (2001).</li><li>Ravid, T., Kreft, S. G., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+soluble+substrates+of+the+Doa10+ubiquitin+ligase+are+degraded+by+distinct+pathways.">Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways.</a> EMBO J. 25, 533-543 (2006).</li><li>Gilon, T., Chomsky, O., Kulka, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signals+for+ubiquitin+system+proteolysis+in+Saccharomyces+cerevisiae.">Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.</a> Embo J. 17, 2759-2766 (1998).</li><li>Hochstrasser, M., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+degradation+of+a+transcriptional+regulator:+the+yeast+2+repressor.">In vivo degradation of a transcriptional regulator: the yeast 2 repressor.</a> Cell. 61, 697-708 (1990).</li><li>Metzger, M. B., Maurer, M. J., Dancy, B. M., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+a+cytosolic+protein+requires+endoplasmic+reticulum-associated+degradation+machinery.">Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery.</a> J Biol Chem. 283, 32302-32316 (2008).</li><li>Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Yeast Genetics and Molecular Biology. 194, (1991).</li><li>Carvalho, P., Goder, V., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ubiquitin-ligase+complexes+define+convergent+pathways+for+the+degradation+of+ER+proteins.">Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.</a> Cell. 126, 361-373 (2006).</li><li>Denic, V., Quan, E. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luminal+surveillance+complex+that+selects+misfolded+glycoproteins+for+ER-associated+degradation.">A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.</a> Cell. 126, 349-359 (2006).</li><li>Wu, P. Y., 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=A+conserved+catalytic+residue+in+the+ubiquitin-conjugating+enzyme+family.">A conserved catalytic residue in the ubiquitin-conjugating enzyme family.</a> Embo J. 22, 5241-5250 (2003).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Fluoroorotic+acid+as+a+selective+agent+in+yeast+molecular+genetics.">5-Fluoroorotic acid as a selective agent in yeast molecular genetics.</a> Methods Enzymol. 154, 164-175 (1987).</li><li>Alfassy, O. S., Cohen, I., Reiss, Y., Tirosh, B., Ravid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placing+a+disrupted+degradation+motif+at+the+C+terminus+of+proteasome+substrates+attenuates+degradation+without+impairing+ubiquitylation.">Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.</a> J Biol Chem. 288, 12645-12653 (2013).</li><li>Cronin, S. R., Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+degradation+with+green+fluorescent+protein.">Measuring protein degradation with green fluorescent protein.</a> Methods in Enzymology. 302, 58-73 (1999).</li></ol>

The authors have nothing to disclose.

在这里，我们描述了基于细胞的生长来确定蛋白质的相对降解率的测定，称为"液体培养基中的生长动力学下的选择性的条件"（GILS）。该GILS法有几个优点:它是简单的设置，数据采集和分析是很简单的，是非常模块化。因此，GILS可以同时应用于多个样品中，可适应于自动化高通量应用的一种用户友好的多井板格式。最重要的是，GILS提供高度可重复的，定量和可靠的数据和比选择为正/负生长的琼?...

泛素 - 蛋白酶体降解系统是一种主要的调节机，它有牵连的蛋白质稳态中的所有真核生物中的维持。 UPS的最初偶联物多泛素分子，以在此之后，多聚遍在蛋白标记的蛋白质被26S蛋白酶体降解的靶蛋白。在大多数情况下，速率限制为泛素介导的降解步骤是泛素化底物，由E2结合酶和E3结扎酶介导的（E3连接酶）1。因此，特定的蛋白质的细胞内稳定性反映了其易泛素缀合和它们的同源泛素酶的活性。 E3连接酶是UPS的主要底物识别元件。因此，这些内切酶识别它们的底物是不存在或是在其稳定的对应2不暴露于降解决定子。例如，许多的细胞周期调节米乌斯进行合成，并为了保持细胞周期进程，以便降低在时间上的具体方式。这些蛋白质的降解往往是由磷酸化控制，通过细胞信号调节的激酶介导的3,4。另一方面，异常折叠的蛋白质是通过隐蔽降解决定子的认可。这些是通常隐藏在天然结构并且在结构扰动被暴露的区域。这种降解决定子包括疏水区5-7和内在的无序段8。 由于泛素系统的精液发现对蛋白质降解和其基本特征中的网织红细胞裂解物9，酵母遗传学是有助的发现许多泛素系统10的组件。酵母作为模式生物的蛋白质降解的由UPS系统的分析的成功主要是由于这样的事实，所述UPS处于高LY保守的所有真核生物4，再加上他们的顺从作为一个实验系统。的确，基于酵母的系统通常用于破译泛素化机械的作用机制。 研究蛋白质降解的生物化学手段，通常需要准备的细胞提取物。虽然动物细胞的蛋白可以在相对 ​​温和的条件，同时保留蛋白质相互作用和功能下被提取，稳健的细胞壁中酵母11的存在需要相当苛刻的中断条件可能影响蛋白的恢复。事实上，不同的程序，酵母细胞破碎有很大的不同在他们恢复完整蛋白质在正确表示它们的相对丰度的细胞数量的能力。此外误差是固有的用于确定特定蛋白质的降解速率的不同方法:代谢标记为基础的"脉冲追踪"的实验，随后通过QQ，MSNmunoprecipitation，以隔离特定蛋白12，往往不是严格的定量。因此，当蛋白质的降解是由该方法相比，格外小心，应解释结果的行使。为了克服这个缺点，另一种放线菌酮（CHX）追法可聘请了12位。在此测定中，翻译抑制剂加入到细胞培养物和蛋白质的稳态水平的时间变化随后被监视。然而，放线菌酮的使用仅限于蛋白质具有相对短的半衰期（&lt;90分钟），由于长期抑制蛋白质的合成是细胞毒性的。值得注意的是，无论是在上述测定中需要使用蛋白质特异性抗体，这并不总是可用的。 为了克服这些技术限制，研究人员已开发了几种方法，不需要的细胞的提取和蛋白质直接处理。一种方法是基于建立营养缺陷型酵母的圣菌株，通过缺失基因编码的必需的代谢酶的获得。这样的基因包括HIS3，LEU2，LYS2和TRP1，对于所需的氨基酸生物合成的酶的编码，以及URA3，其编码的OMP脱羧酶（的Ura3）的嘧啶核糖核苷酸生物合成的必需酶。的ura3已被广泛用于蛋白质降解研究。在这些测定中，的Ura3的组成型表达拯救的ura3细胞的尿嘧啶缺陷型培养基中生长13。因此，通过降解决定子的融合不稳定的Ura3可以减少对基本培养基中缺乏尿嘧啶的细胞生长。此方法已被应用于各种蛋白质降解的研究，包括降解的识别决定因素5，E3连接酶14和辅助泛素化因子15和新颖的UPS降解决定子16的发现。所有的这些方法中使用的琼脂平板测定法读出的细胞生长。然而，T他生长标准（正/负增长），而健壮和有效的，主要是定性的，并且不提供用于评估降解决定子的效力或各种辅助降解因素的相对贡献重要的定量信息。 因此，我们开发和利用酵母载体和筛选方法的URA3 -degron融合系统使蛋白质降解系统和定量的分析。该协议基于一个易于手柄测定，其测量的生长动力学在选择性条件下（GILS）液体培养和对标准生长曲线的生成。酵母生长动力学的特征在于三个主要阶段 - 滞后，指数（日志）和固定相。计算过程中的选择性的条件下的对数期，这是由的Ura3，降解决定子的表达水平的测定酵母的复制动力学，提供了一个无偏定量测定öF蛋白的降解。这个方法可以适用于测量和比较同时在多种菌株和在各种条件下的多个UPS基材的降解速率。

<table><tbody><tr><td>Difco yeast nitrogen base w/o amino acids and ammonium sulfate</td><td>BD Biosciences</td><td>233520</td><td>For yeast growth on SD minimal media.&amp;nbsp; Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan</td></tr><tr><td>Ammonium sulfate</td><td>Sigma - Aldrich</td><td>A4418</td><td></td></tr><tr><td>Glucose</td><td>Sigma - Aldrich</td><td>16325</td><td></td></tr><tr><td>Adenine</td><td>Sigma - Aldrich</td><td>A8626</td><td></td></tr><tr><td>Uracil</td><td>Sigma - Aldrich</td><td>U0750</td><td></td></tr><tr><td>Amino acids</td><td>Highest purity available&amp;nbsp;</td><td></td><td></td></tr><tr><td>96-well plates</td><td>Nunc</td><td>167008</td><td>Any other compatible brand can be used</td></tr><tr><td>Cyclohexamide&amp;nbsp;</td><td>Sigma - Aldrich</td><td>C7698</td><td>Working conc. 0.5 mg/ml</td></tr><tr><td>Infinite 200 PRO series</td><td>Tecan</td><td></td><td>For yeast incubation and OD&lt;sub&gt;600&lt;/sub&gt; measurements. Any other compatible temp-controled reader can be used.</td></tr><tr><td>MDTcalc</td><td></td><td></td><td>Experimental software</td></tr></tbody></table>

reporter-based growth assay for systematic analysis of protein degradation

通过泛素 - 蛋白酶体系统（UPS），蛋白质降解是一个主要的调节机制在所有真核生物蛋白的动态平衡。该标准方法测定细胞内蛋白质降解依赖于生化分析了以下的蛋白质下降的动力学。这样的方法通常是费力和费时，因此不适合进行实验，目的是评估多种底物和降解的条件。作为另一种选择，细胞生长为基础的分析已被开发出来，那是，在他们的常规格式，不能定量确定在蛋白质水平的相对变化终点分析。 在这里，我们描述了一种方法，通过它们连接到酵母细胞的生长动力学，忠实地决定改变蛋白质的降解率。该方法是基于已建立的选择系统，其中的URA3的尿嘧啶营养缺陷型-deleted酵母细胞是通过外源表达报告蛋白获救，包括必要的URA3基因和一个退化行列式（降解决定子）之间的融合。报告蛋白被设计成使得它的合成速率是恒定的，而其降解速率由降解决定子确定。作为细胞生长的尿嘧啶缺陷型培养基是成比例的Ura3的相对水平，生长动力学的完全依赖于报告蛋白降解。 这种方法精确测量变化的细胞内蛋白质降解动力学。它适用于:（a）评估称为泛素结合因子蛋白水解（B）的相对贡献E2结合酶结构与功能分析（三）鉴定及新型降解决定子表征。该degron- URA3基于系统的应用超越蛋白降解字段，因为它也可以适用于蛋白质水平与其他细胞途径功能相关联的监视更改。

调查记者衬底的降解Doa10途径的酶的作用 为了测试GILS方法与其相比较传统的降解测定法的有效性。该实验评估对ER-膜的组成部分的相对贡献本地化Doa10的E3连接酶复合物20,21的蛋白质量控制记者基板Deg1- VU（ 图3A）的降解。该Deg1- VU质粒整合到野生型细胞或进入细胞缺乏该基因的E2结合酶，UBC7（ubc7）和蛋白质的稳定?...

Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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 ...

reporter-based-growth-assay-for-systematic-analysis-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.6K 次观看.  The Hebrew University of Jerusalem. 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.

视频: Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

科研

发育生物学

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

癌症研究

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

生物化学

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

生物学

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 ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

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 ...

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.

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 ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

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://appjove.unicartagenaproxy.elogim.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 ...

记者的增长分析的蛋白质降解的系统分析

摘要

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此视频中的章节

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

测定蛋白酶体降解的植物无细胞系统

在培养的哺乳动物细胞大规模并行报告基因分析

成长型测定和遗传需求生化确认的蛋白质降解

测定法错误折叠的蛋白质在细胞中的降解

卡诺哈布迪蒂斯伊利根斯新蛋白合成率评估

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

记者的增长分析的蛋白质降解的系统分析

摘要

探索更多视频

此视频中的章节

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

测定蛋白酶体降解的植物无细胞系统

在培养的哺乳动物细胞大规模并行报告基因分析

成长型测定和遗传需求生化确认的蛋白质降解

测定法错误折叠的蛋白质在细胞中的降解

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

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

卡诺哈布迪蒂斯伊利根斯新蛋白合成率评估