该莱夫科夫斯基家庭基金会/ Liz和埃里克·莱夫科夫斯基创新研究奖，以AV我们想 - 这里介绍的项目是由美国国立卫生研究院从/ NCI（NCI-R01CA182506-01A1）的资助，以及由罗伯特·卢里综合癌症中心的支持要感谢批判性阅读和编辑这篇稿子的实验室成员（卡罗尔·奥卡拉汉和伊丽莎白安妮·韦恩机场）。

1. 在体外试验脱乙酰<ol><li> SIRT2纯化<ol><li>准备在8ml含10％FBS和抗生素的培养，并在37℃，5％CO 2的组织培养箱中生长至约70％汇合，第二天的HEK 293T细胞的10cm的培养皿。 </li><li>第二天，共转染4微克PCDH-普罗-GFP-SIRT2-标志（在我们的实验室制成慢病毒），4微克pCMV- dR8.2 DVPR（包装载体）和0.5微克的pCMV-VSV-G（包络线矢量） 18使用聚乙烯亚胺（PEI）在3微升的PEI /微克DNA的一比值。 </li><li>经过12小时更换介质。 </li><li>后36-48小时收集上清。 </li><li>离心1000 XG 5分钟，4℃清除杂物。 </li><li>通过0.22微米的过滤器过滤后，使SIRT2慢病毒的1ml等份，并保持在-80℃。 </li><li>在冰上解冻SIRT2慢病毒（对病毒储存在-80℃下是很重要的，当不使用病毒）。 </li><li>感染80-90％汇合的HEK 293T细胞的6孔平板，在8微克/毫升聚凝胺的具有1毫升的慢病毒。 </li><li>放置在37℃培养箱中病毒感染的细胞，直到第二天（24小时）。 </li><li> 24小时后更换培养基。 </li><li>感染后48小时，通过在荧光显微镜下检测GFP阳性细胞检查感染效率。 </li><li>如果感染效率是好的（至少40-50％感染的细胞），含有2微克/毫升嘌呤霉素的培养基中过表达的细胞，选择稳定SIRT2更换介质。 注意:这大约需要2周，并且将培养基每3-4天更换一次。非感染细胞的死亡在这个选择期间，只感染细胞的慢病毒SIRT2增长。 </li><li>在10％SDS-PAGE凝胶上运行40微克总蛋白后:（1,000稀释1）在开始PURIF之前通过Western印迹使用抗Flag抗体分析Flag标记SIRT2的表达ication过程（推荐）19,20。 </li><li>净化活性SIRT2，分裂细胞以1:3的比例通过胰蛋白酶消化，以便有HEK 293T细胞稳定过表达的Flag-SIRT2的足够菜（十10厘米菜将使随后的实验够蛋白的纯化）。 <ol><li>到trypsinize的细胞，除去培养基，并通过漂洗细胞，用无菌DPBS消除残余的血清。慢慢加入2.5 0.25％胰蛋白酶-EDTA溶液中，以覆盖细胞单层，在室温下孵育30-45秒。除去胰蛋白酶-EDTA溶液后，孵育在37℃，直至细胞开始分离。再加入培养液和细胞转移到新菜。 </li></ol></li><li>除去培养基，在4℃下以1000 xg离心在PBS洗涤细胞两次，并收集通过胰蛋白酶的细胞，然后离心10分钟。 </li><li>于500μl裂解缓冲液A（20mM的HEPES pH值7.9，18毫米氯化钾，0.2mM的EGTA，1.5mM的MgCl 2的裂解细胞沉淀，20％甘油，0.1％NP-40），其包括蛋白酶抑制剂鸡尾酒和下恒定旋转1μM的TSA的30分钟，在4℃。 </li><li>离心机在14000 xg离心在4℃下15分钟，并转移上清液至新管。 </li><li>使用缀合，如下所述琼脂糖珠粒的抗Flag抗体进行免疫沉淀。 <ol><li>添加200-300微升偶联至琼脂糖珠的蛋白质裂解物（10-20毫克总蛋白）抗Flag抗体的。 </li><li>在4℃下以恒定的搅拌温育过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下5分钟。 </li><li>洗涤沉淀5次，用10ml裂解缓冲液A以1000 xg离心在4℃下5分钟。每次洗涤后，沉淀珠子和替换用缓冲液A将上清液</li></ol></li><li>制备1×FLAG肽溶液（包括蛋白酶抑制剂鸡尾酒和1μM的TSA在PBS中）。 </li><li>加入500μl1X标志肽类的Ë溶液到琼脂糖珠，并在4℃下恒定旋转温育30分钟。 </li><li>收集离心上清在6000 xg离心在4℃下2分钟。 </li><li>重复前两个步骤。 </li><li>通过使用过滤管和离心15,000 XG 1分钟，4℃卸下上清珠。 </li><li>浓缩洗脱样品，直到最终蛋白质浓度为1微克/微升使用超滤膜。 </li><li>通过使用洗脱样品的1微克进行电泳检查纯化过程。 </li><li>用市售的染色液染色凝胶来确认SIRT2纯化。 </li><li>保持纯净SIRT2在-80℃。 </li></ol></li><li>乙酰化的蛋白质 - 底物的纯化<ol><li>准备好10厘米×10厘米的培养皿与HEK 293T细胞为约70％汇合的第二天。 </li><li>第二天，共转染细胞与标记的5微克标记质粒载体expressin克蛋白质 - 底物以及5微克特定乙酰转移可在3微升的PEI /微克DNA的一比率乙酰使用的PEI的蛋白质。 注意:如果特定乙酰转移是未知的，共转染与已知的组蛋白乙酰基转移酶（帽）如P300，CBP，GCN5，Tip60的和PCAF的混合物。 </li><li>经过12小时更换培养基。 </li><li>当天裂解细胞前，治疗用1μMTSA和2mM烟酰胺（NAM）过夜的细胞通过更换用含TSA / NAM通过抑制脱乙酰酶最大化蛋白质 - 底物的乙酰化水平的介质的培养基中。 </li><li>转染后48小时，去除培养基，在PBS中通过胰蛋白酶消化，随后离心以1000 xg离心在4℃下洗涤细胞两次，并收集细胞10分钟。 </li><li>在每皿加入500μl裂解缓冲液A裂解细胞沉淀液（20mM HEPES pH值7.9，18毫米氯化钾，0.2mM的EGTA，1.5mM的氯化镁，20％甘油，0.1％NP-40），其包括蛋白酶Inhibitors鸡尾酒，1μM的TSA和在4℃下恒定旋转2mM的NAM 30分钟。 </li><li>离心机在14000 xg离心在4℃下15分钟，并转移上清液至新管。 </li><li>使用缀合，如下所述琼脂糖珠粒的抗Flag抗体进行免疫沉淀。 <ol><li>添加200-300微升偶联至琼脂糖珠的蛋白质裂解物（10-20毫克总蛋白）抗Flag抗体的。 </li><li>在4℃下以恒定的搅拌温育过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下5分钟。 </li><li>洗涤沉淀2次，用10ml裂解缓冲液A以1000 xg离心在4℃下5分钟。每次洗涤后，沉淀珠子和替换用裂解缓冲液A中的上清</li><li>洗涤沉淀，用10ml裂解缓冲液A 3次无NAM在1000×g离心5分钟，4℃。每次洗涤后，沉淀珠子和替换用裂解缓冲液A.上清液它性重要NT不携带在任何不结盟运动的脱乙酰反应。 </li></ol></li><li>制备1×FLAG肽溶液（包括蛋白酶抑制剂和1μM的TSA在PBS中）。 </li><li>加入500微升1×FLAG肽溶液至琼脂糖珠孵育下恒定旋转，在4℃，30分钟。 </li><li>收集离心上清在6000 xg离心在4℃下2分钟。 </li><li>重复前两个步骤。 </li><li>通过使用过滤管和离心15,000 XG 1分钟，4℃卸下上清珠。 </li><li>使用超滤膜，浓缩洗脱样品，直至蛋白质浓度为1微克/微升。 </li><li>通过在4-12％SDS-PAGE凝胶上运行1微升洗脱的样品进行电泳。 </li><li> （:1000稀释1）和蛋白质底物（稀释取决于所用的特异性抗体）使用针对交流-K抗体western印迹确认蛋白 - 底物的乙酰化。 </li><li>保持普里田间在-80℃下的乙酰化蛋白 - 底物。 </li></ol></li><li> 体外脱乙酰反应<ol><li>制备脱乙酰缓冲液B（50mM的Tris-盐酸，pH为7.5，150 mM氯化钠，1mM的MgCl 2的0.5微米，TSA） </li><li>准备在不同管3不同的反应在20μl的最终体积（ 见表1）。 </li><li>包括额外的反应由SIRT2（可选）确认蛋白底物脱乙酰化。添加纯化的脱乙酰无效突变SIRT2 2微克到反应，而不是脱乙酰活性SIRT2（这可以使用在1.1中描述的协议来完成使用PCDH-普罗-GFP-SIRT2 H187Y -flag慢病毒后），或添加的10mM烟酰胺（NAM ）反应以抑制SIRT2的脱乙酰酶活性（ 表1）。 </li><li>孵育反应为在30℃下持续搅拌3小时。 </li><li>通过加入20微升2×样品缓冲液终止反应，煮沸样品F或在95℃下10分钟。 </li><li>通过运行在4-12％SDS-PAGE凝胶的反应混合物进行电泳和使用抗AC-κ ​​抗体（1:1000稀释）检测由印迹蛋白质-底物的乙酰化状态19,20。 </li></ol></li></ol> 2. 在体内脱乙酰含量<ol><li> SIRT2表达在培养的细胞<ol><li>准备与HEK 293T细胞10厘米培养皿稳定过表达PCDH-普罗-GFP-空载体，PCDH-普罗-GFP-SIRT2-旗及PCDH-普罗-GFP-SIRT2 H187Y -flag（如在1.1中描述）为约70 ％汇合。 </li><li>另外，准备10厘米培养皿与细胞约70％汇合，并使用上述在3微升PEI /微克DNA比使用PEI提到的载体的5微克执行瞬时表达。 </li><li>执行使用抗Flag抗体总细胞提取物的蛋白质印迹（1:1000稀释）来演示的标示 - 叔过表达agged SIRT2 19。 </li></ol></li><li> RNA干扰拦截SIRT2在培养的细胞<ol><li>制备的HEK 293T细胞的10cm的培养皿为约70％汇合的第二天。 </li><li>第二天，共转染4微克pLKO1-普罗-SH SIRT2（慢病毒载体）19，4微克pCMV- dR8.2 DVPR（包装载体）和0.5微克的pCMV-VSV-G（包络线矢量）18使用聚乙烯亚胺（PEI）在3微升的PEI /微克DNA的一比值。 注意:对于撞倒SIRT2，我们用简单的发夹的shRNA由RNAi协会（TRC）设计了pLKO.1慢病毒载体。 </li><li>操作与1.1中描述的生产shSIRT2慢病毒并感染靶细胞拦截SIRT2。 </li><li> （:1000稀释1）在10％SDS-PAGE凝胶上运行总蛋白的40微克后表现出有效的SIRT2敲低使用抗SIRT2抗体进行总细胞提取物的Western印迹。 </li><li>此外，与细胞约70％汇合准备10厘米培养皿和使用以下制造商的说明的siRNA进行SIRT2短暂击倒。 </li><li> （:1000稀释1）在10％SDS-PAGE凝胶上运行总蛋白的40微克后表现出有效的SIRT2敲低使用抗SIRT2抗体进行总细胞提取物的Western印迹。 </li></ol></li><li>免疫沉淀和免疫印迹检测培养细胞乙酰化水平<ol><li>从任一细胞过度SIRT2收集细胞沉淀之前12小时（见2.1以上）或SIRT2撞倒的细胞（见上文2.2），添加1微米的TSA到培养基中，以抑制非III类组蛋白脱乙酰。 </li><li>除去培养基，在4℃下以1000 xg离心在PBS洗涤细胞两次，并收集通过胰蛋白酶的细胞，然后离心10分钟。 </li><li>加入10 mL裂解液A（20毫米的HEPES pH值7.9，18毫米氯化钾，0.2毫米EGTA，1.5毫MgCl 2的，20％甘油，0.1％NP-40），其包括蛋白酶抑制剂鸡尾酒，1μM的TSA和2mM NAM。 </li><li>孵育在4℃下恒定旋转30分钟。 </li><li>离心收集上清在20000 XG在4°C 15分钟。 </li><li>通过使用Bradford蛋白测定计算蛋白质浓度。 </li><li>转印1mg总蛋白质的新管中的使用裂解缓冲液A.使用表2作为参考1ml的最终体积，以从细胞中任一过表达SIRT2或SIRT2击倒后制备蛋白质样品。 </li><li>执行使用针对特定的蛋白质 - 底物的抗体与蛋白A / G一起免疫沉淀（建议将40μl珠浆料）。或者，使用偶联到琼脂糖珠防AC-κ抗体（20μL珠浆通常是不够的）免疫沉淀所有的蛋白质乙酰化。 </li><li>在根据CONSTA 4℃孵育样本NT旋转过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下2分钟。 </li><li>在4℃下洗珠5次用1毫升缓冲液A以1000 xg离心2分钟。 </li><li>珠悬浮在40μl的2X样品缓冲液。 </li><li>煮沸样品在95℃下10分钟。 </li><li>通过运行在4-12％SDS-PAGE凝胶洗脱的样品（从步骤2.3.12 40微升），而不会干扰珠粒在传送洗脱的样品进行电泳。 </li><li>使用抗AC-κ抗体通过Western印迹法检测蛋白质 - 底物的乙酰化水平（1:1,000稀释度），如果是用于免疫沉淀的蛋白质 - 底物特异性抗体，或针对该蛋白 - 底物的特异性抗体（遵循建议特异性抗体对稀释）如果被用于免疫沉淀的抗徒-K珠。 </li></ol></li></ol>

<ol>
	<li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+age+of+crosstalk:+phosphorylation,+ubiquitination,+and+beyond.">The age of crosstalk: phosphorylation, ubiquitination, and beyond.</a> Molecular Cell. 28, 730-738 (2007).</li><li>Gao, M., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+regulators:+control+of+protein+ubiquitination+and+ubiquitin-like+modifications+by+extracellular+stimuli.">Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli.</a> Molecular Cell. 19, 581-593 (2005).</li><li>Philp, A., Rowland, T., Perez-Schindler, J., Schenk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+acetylome:+translating+targeted+proteomics+into+meaningful+physiology.">Understanding the acetylome: translating targeted proteomics into meaningful physiology.</a> American Journal of Physiology. Cell Physiology. 307, C763-C773 (2014).</li><li>Allfrey, V. G., Faulkner, R., Mirsky, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+Methylation+of+Histones+and+Their+Possible+Role+in+the+Regulation+of+Rna+Synthesis.">Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 51, 786-794 (1964).</li><li>Shahbazian, M. D., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+site-specific+histone+acetylation+and+deacetylation.">Functions of site-specific histone acetylation and deacetylation.</a> Annual Review of Biochemistry. 76, 75-100 (2007).</li><li>L'Hernault, S. W., Rosenbaum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+alpha-tubulin+is+posttranslationally+modified+in+the+flagella+during+flagellar+assembly.">Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly.</a> The Journal of Cell Biology. 97, 258-263 (1983).</li><li>Kim, S. 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=Substrate+and+functional+diversity+of+lysine+acetylation+revealed+by+a+proteomics+survey.">Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.</a> Molecular Cell. 23, 607-618 (2006).</li><li>Choudhary, 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=Lysine+acetylation+targets+protein+complexes+and+co-regulates+major+cellular+functions.">Lysine acetylation targets protein complexes and co-regulates major cellular functions.</a> Science. 325, 834-840 (2009).</li><li>Smith, K. T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+the+acetylome.">Introducing the acetylome.</a> Nature Biotechnology. 27, 917-919 (2009).</li><li>Roth, S. Y., Denu, J. M., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetyltransferases.">Histone acetyltransferases.</a> Annual Review of Biochemistry. 70, 81-120 (2001).</li><li>Ozden, O., 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=Acetylation+of+MnSOD+directs+enzymatic+activity+responding+to+cellular+nutrient+status+or+oxidative+stress.">Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress.</a> Aging. 3, 102-107 (2011).</li><li>Anderson, K. A., Hirschey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+acetylation+regulates+metabolism.">Mitochondrial protein acetylation regulates metabolism.</a> Essays in Biochemistry. 52, 23-35 (2012).</li><li>Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+lysine+acetylation+controls+the+activity+of+the+mitochondrial+enzyme+acetyl-CoA+synthetase+2.">Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10224-10229 (2006).</li><li>Haberland, M., Montgomery, R. L., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+histone+deacetylases+in+development+and+physiology:+implications+for+disease+and+therapy.">The many roles of histone deacetylases in development and physiology: implications for disease and therapy.</a> Nature Reviews. Genetics. 10, 32-42 (2009).</li><li>Finkel, T., Deng, C. X., Mostoslavsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+biology+and+physiology+of+sirtuins.">Recent progress in the biology and physiology of sirtuins.</a> Nature. 460, 587-591 (2009).</li><li>Houtkooper, R. H., Pirinen, E., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+as+regulators+of+metabolism+and+healthspan.">Sirtuins as regulators of metabolism and healthspan.</a> Nature reviews. Molecular Cell Biology. 13, 225-238 (2012).</li><li>Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+landscape+of+lysine+acetylation+links+metabolism+and+cell+signalling.">The growing landscape of lysine acetylation links metabolism and cell signalling.</a> Nature Reviews. Molecular Cell Biology. 15, 536-550 (2014).</li><li>Stewart, S. A., 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=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9, 493-501 (2003).</li><li>Kim, H. S., 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=SIRT2+maintains+genome+integrity+and+suppresses+tumorigenesis+through+regulating+APC/C+activity.">SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.</a> Cancer Cell. 20, 487-499 (2011).</li><li>Zhang, H., 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=SIRT2+directs+the+replication+stress+response+through+CDK9+deacetylation.">SIRT2 directs the replication stress response through CDK9 deacetylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 13546-13551 (2013).</li><li>Weinert, B. T., 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=Proteome-wide+mapping+of+the+Drosophila+acetylome+demonstrates+a+high+degree+of+conservation+of+lysine+acetylation.">Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.</a> Science Signaling. 4, ra48 (2011).</li><li>Still, A. J., 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=Quantification+of+mitochondrial+acetylation+dynamics+highlights+prominent+sites+of+metabolic+regulation.">Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.</a> The Journal of Biological Chemistry. 288, 26209-26219 (2013).</li><li>Sadoul, K., Wang, J., Diagouraga, B., Khochbin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tale+of+protein+lysine+acetylation+in+the+cytoplasm.">The tale of protein lysine acetylation in the cytoplasm.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 970382 (2011).</li><li>Preble, J. M., 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=Rapid+isolation+and+purification+of+mitochondria+for+transplantation+by+tissue+dissociation+and+differential+filtration.">Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.</a> Journal of Visualized Experiments : JoVE. , e51682 (2014).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sirtuins:+regulators+of+protein+acylation+and+metabolism.">Mitochondrial sirtuins: regulators of protein acylation and metabolism.</a> Trends in Endocrinology and Metabolism: TEM. 23, 467-476 (2012).</li></ol>

作者什么都没有透露。

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in the future, highlighting the importance of reversible acetylation as a regulatory modification directing diverse cellular processes. Regardless of the magnitude of acetylation, identifying the deacetylases which can specifically deacetylate such protein-substrates is an important first step in unraveling the biology of protein acetylation and understanding the complexity of this phenomenon. Towards this direction, use of both in vitro and in vivo deacetylation assays are crucial and necessary to establish the regulatory interplay between deacetylases and their substrates.
Here we present an example of how deacetylation assays can be used in order for an acetylated protein to be considered as a legitimate deacetylation target of SIRT2. As it has already been mentioned, the sirtuin family of proteins consists of seven members which can be found in the cytoplasm, mitochondria, and nucleus 15. Given the increasing interest regarding the role of these proteins in the different subcellular compartments which can be associated with distinct cellular processes, the above described deacetylation assays can be used with slight modifications to identify new substrates for all different members of the sirtuin family. For example, when mitochondrial proteins are used in deacetylation assays related to SIRT3, it may be necessary to include a mitochondrial isolation step 24,25 between cell lysis and immunoprecipitation during the purification process for the in vitro deacetylation assay or during the in vivo deacetylation assay before detecting acetylated protein levels. In a similar way, when nuclear proteins need to be used as deacetylation targets in these assays, nuclear extracts following subcellular fractionation might be used for protein purification and the in vivo deacetylation assay. 
A critical step for executing the in vitro deacetylation assay is the successful purification of both the deacetylase and the acetylated protein-substrate. Verification of the cells overexpressing the deacetylase as well as the acetylated protein-substrate before the initiation of the purification step is highly recommended. Furthermore, detection of the purified proteins after running a small amount of the isolated proteins on an SDS-PAGE gel before the in vitro deacetylation reaction is also recommended. This will not only confirm the completion of the purification step but will help investigators to focus their efforts on optimizing the deacetylation reaction conditions for any given protein-substrate and deacetylase of interest in case of an unsuccessful deacetylation assay which may require changes in the enzyme-substrate ratio used. Of note, even if a positive deacetylation reaction is detected using the in vitro assay, further validation is required before any conclusion can be made regarding the establishment of a protein as a deacetylation target of a specific deacetylase. This limitation is due to the fact that the co-presence of the substrate and the deacetylase in the tube does not mean that both can be found in the same complex in cells or tissues. This implies that the deacetylation needs to be further validated in vivo. With the focus on the in vivo deacetylation assay, it is worth mentioning that the steady state acetylation levels of a given protein under the experimental conditions tested is a crucial factor in determining whether overexpression or knockdown experiments are more suitable to establish the functional interplay between a deacetylase and its substrate. High levels of acetylation may direct the experimental design towards overexpressing the specific deacetylase to detect a decrease in the acetylation levels. On the contrary, when low levels of acetylation are detected under normal conditions, increased acetylation after knocking down the specific deacetylase might establish the mechanistic connection between a deacetylase and its substrate. Taking all these together, it is clear that the deacetylation assays described in this protocol are necessary to link a deacetylase with its potential substrate.
The next challenges in the field of lysine acetylation dictate that deacetylation assays will be used in the future to address some of the missing links. The considerable gap between the large number of identified mitochondrial lysine sites and the few with a validated regulatory function 26 highlights the need to distinguish bona fide functional acetylation sites from what may be widespread spurious modifications. In vitro and in vivo deacetylation assays carried over by specific deacetylases may be further combined with small scale MS analysis to reveal specific target lysines on a tested substrate to distinguish functional acetylation sites from what may be non-regulatory acetylated lysines. In this regard, sites which are deacetylated due to the enzymatic activity of a sirtuin are more likely to play a functional role compared to sites that are not deacetylated. Another significant challenge is the examination of lysine acetylation in a physiological context. It seems very likely that the acetylation profile of a cell may vary under specific experimental conditions. In vivo deacetylation assays in cells under specific experimental conditions can provide further insight into the connection between the deacetylase and the potential substrate in physiological and pathophysiological scenarios depending on the cell type, or the exposure to specific signaling molecules and stressors. Such studies can establish the functional role of a deacetylation event in a physiological context and connect it to a phenotypic outcome or response. More importantly, deacetylation assays in combination with small scale MS analysis can further provide meaningful information regarding regulatory acetylated lysines under specific cellular conditions.
In conclusion, identification of the enzymes that deacetylate acetylated proteins and sites by using both in vitro and in vivo deacetylation assays will help to resolve the complexity of the acetylome and will contribute to better understanding of the regulatory role of acetylation under various physiological conditions.

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table><tbody><tr><td>cell culture dishes</td><td>Denville Scientific Inc.</td><td>T1110 and T1115</td><td></td></tr><tr><td>pCDH-puro-GFP lentiviral vector</td><td>System Biosciences</td><td>CD513B-1</td><td></td></tr><tr><td>pCMV- dR8.2 dvpr (packaging vector)</td><td>Addgene</td><td>8455</td><td></td></tr><tr><td>pCMV-VSV-G (envelope vector)</td><td>Addgene</td><td>8454</td><td></td></tr><tr><td>polyethylenimine (PEI)&amp;nbsp;</td><td>Polysciences Inc.</td><td>24885</td><td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td></tr><tr><td>0.22 &amp;mu;m filters</td><td>Denville Scientific Inc.</td><td>F5512</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>H9268</td><td></td></tr><tr><td>fluorescent microscope</td><td>Carl Zeiss MicroImaging Inc.</td><td>Axiovert 200</td><td></td></tr><tr><td>puromycin</td><td>Invivogen</td><td>A11138-03</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>anti-Flag antibody</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>34596</td><td></td></tr><tr><td>protease inhibitors coctail 100x</td><td>Biotool</td><td>B14001</td><td></td></tr><tr><td>Trichostatin A (TSA)</td><td>Sigma</td><td>T8552</td><td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class III HDACs (sirtuins)</td></tr><tr><td>anti-Flag agarose beads</td><td>Sigma</td><td>A2220</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>rotator</td><td>Thermo Scientific</td><td>415220Q</td><td></td></tr><tr><td>filter tubes</td><td>Millipore</td><td>UFC30HV00</td><td></td></tr><tr><td>Flag peptide&amp;nbsp;</td><td>Sigma</td><td>F3290</td><td></td></tr><tr><td>Vivaspin Centrifugal Concentrator</td><td>Sartorius Stedim Biotech S.A.</td><td>VS0102</td><td></td></tr><tr><td>SimplyBlue SafeStain solution&amp;nbsp;</td><td>Invitrogen</td><td>LC6060</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Techologies</td><td>NP0007</td><td></td></tr><tr><td>Tris-HCl pH 7.5</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>NAD&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma</td><td>N0632</td><td>required cofactor for sirtuins</td></tr><tr><td>nicotinamide (NAM)</td><td>Sigma</td><td>72340</td><td>selective inhibitor class III HDACs (sirtuins)</td></tr><tr><td>pLKO.1 lentiviral vector&amp;nbsp;</td><td>Addgene</td><td>8453</td><td></td></tr><tr><td>SIRT2 si RNA&amp;nbsp;</td><td>Qiagen</td><td>GS22933</td><td></td></tr><tr><td>anti-SIRT2 antibody</td><td>Proteintech</td><td>15345-1-AP</td><td></td></tr><tr><td>Bradford protein assay</td><td>BIO-RAD</td><td>500-0006</td><td></td></tr><tr><td>anti Ac-K agarose beads</td><td>Immunechem</td><td>ICP0388</td><td></td></tr></tbody></table>

deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

为了使蛋白质被认为是用于与脱乙酰酶活性的任何酶合法脱乙酰目标，无论是在体外和体内脱乙酰测定需要执行以建立脱乙酰酶和其底物之间的相互作用。 用于体外脱乙酰酶测定中，可以进行该测定之前，需要的脱乙酰酶和乙酰化蛋白底物两者的纯化。在这里，我们使用作为要研究的特定脱乙酰酶细胞质沉默调节蛋白SIRT2。 SIRT2是强烈建议作为用于脱乙酰测定的阴性对照一个脱乙酰酶和使用它缺乏脱乙酰酶活性的突变体的。使用在1.1中描述的纯化过程，既SIRT2和SIRT2 H187Y可以成功地在1微克/微升的最终浓度进行纯化。这可以在一个凝胶随后染色运行纯化的蛋白质后的确认（ 图1A，上图），以及使用抗Flag抗体Western印迹后因为两者SIRT2和SIRT2 H187Y的标签带标示的肽（ 图2A，下）。相同的纯化步骤可以遵循为具有一些修改的蛋白底物的纯化。蛋白质需要它可用于脱乙酰测定，这意味着它需要在细胞过度具体乙酰转移或能够乙酰化的蛋白帽子的混合物被纯化之前将乙酰化。使用在1.2中描述的纯化过程中，乙酰化蛋白质可纯化（ 图1B，上图）。以确认该蛋白确实乙酰并且可用于在体外脱乙酰化测定中，使用抗AC-κ ​​抗体Western印迹是必要显示在细胞中过表达乙酰基转移（ 图1B中的纯化的蛋白质的增加的乙酰化水平，罗威r）的。 成功的蛋白质纯化后，可以进行体外脱乙酰化测定法。乙酰化酶，包括SIRT2，是III类组蛋白去乙酰化酶的赖氨酸，因为他们需要NAD +为他们的酶功能，与其他脱乙酰基酶是不同的。与此相一致，没有脱乙酰可以通过Western印迹使用抗AC-κ ​​抗体在不存在NAD +的检测，不论催化剂活性SIRT2的存在（ 图2，泳道2比1）。相反，降低了乙酰化蛋白的乙酰化水平时既SIRT2和NAD +的存在于反应表明，该蛋白质可以被认为是对于SIRT2一个脱乙酰目标。为了验证这些结果，可以使用不同的阴性对照。例如，在蛋白质的脱乙酰水平没有差异可以被检测，当脱乙酰缺陷SIRT2（SIRT2 H187Y）被用来代替具有催化活性的野生型SIRT2（ 图2，泳道5比4）。当一个完善的沉默调节蛋白的抑制剂，NAM，加入到反应混合物中，这意味着在该蛋白的乙酰化水平的降低是由SIRT2的脱乙酰酶活性介导的类似的效果就可以看出。 异常脱乙酰参与几个细胞过程和年龄相关的疾病。因此，在深化对脱乙酰酶和其底物之间的相互作用的知识的一个关键步骤是建立这个互联互通细胞，其中这种现象可能有生理方面的影响。此外， 在体内检查在细胞培养系统中的脱乙酰允许在其中可以更容易地连接到特定表型或结果的细胞或组织特定上下文脱乙酰事件的研究。这不包括在体外 DEAC所检测的可能性etylation活性人工由于脱乙酰酶和其在管的同时基板，两者的存在下可能永远不会在细胞的正常生理条件下发生的。为在体内脱乙酰测定中，细胞过度既SIRT2和SIRT2 H187Y以及蛋白底物可以按照在2.1和2.2中描述的方法被使用。从这些细胞中制备细胞提取物时，可以确认表达SIRT2，SIRT2 H187Y，并且使用特异性抗体通过蛋白质印迹蛋白底物。在这里所描述的，测定的所有外源性表达的蛋白质的标签，便于在进行的实验（ 图3，下图，以检测HA标记的SIRT2 / SIRT2 H187Y和中间面板以检测Flag标记蛋白底物）。确定目标蛋白是否是SIRT2的脱乙酰基片，用抗Flag抗体拉下靶蛋白f被进行免疫沉淀使用抗AC-κ抗体通过Western印迹ollowed。减少蛋白质的乙酰化水平在细胞中表达SIRT2（ 图3上图，泳道3比2）和未能影响细胞乙酰水平表达脱乙酰酶缺乏SIRT2突变体（ 图3上面板，泳道4比3）表明乙酰化的蛋白质可以是在体内特定脱乙酰酶的脱乙酰目标。当SIRT2介导的脱乙酰是在所研究的细胞用NAM处理细胞（ 图3上面板，泳道5比3）建立特定脱乙酰酶-底轴之后禁止这可以被进一步证实。 <img alt="图1" src="/files/ftp_upload/53563/53563fig1.jpg" /> 图1:脱乙酰酶和乙酰化蛋白-底物两者的纯化，以用于 体外 脱乙酰化测定（A）既SIRT2和SIRT2 H187Y（脱乙酰缺陷突变体）的1微克被上按照如在1.1中描述的纯化方案的凝胶运行。将凝胶用市售染色液和脱色后染色，既可以检测纯化的蛋白质（上部）。蛋白质可以使用​​抗Flag抗体（低级）在PVDF膜和免疫印迹转印后进行检测。 （B）的蛋白底物和超乙酰化蛋白底物的1微克（的α-烯醇酶被用作基于在我们的实验室中产生质谱未公布的数据的SIRT2衬底）如1.2所描述的以下的纯化方案的凝胶进行运行。将凝胶用市售染色溶液和脱色后，纯化的蛋白质底物可被检测（上部）。乙酰化可使用抗AC-κ抗体（中）在PVDF膜和免疫印迹转印后进行检测。总磷<html> roteins可以使用抗Flag抗体（下）被检测到。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53563/53563fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53563/53563fig2.jpg" /> 图2: 在体外 脱乙酰化测定法将纯化的乙酰化蛋白底物（的α-烯醇酶） ​​与SIRT2或SIRT2 H187Y（脱乙酰缺陷突变体）在NAD +的存在下培养（泳道4和5）。降低乙酰水平通过Western印迹使用抗AC-κ ​​抗体在SIRT2的存在下，但不是在SIRT2 H187Y的存在（泳道4比5）检测到。当在反应混合物中不包含NAD +被没有观察到脱乙酰酶活性（泳道2比4），或当NAM被加入到反应混合物（泳道6对4）。://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53563/53563fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53563/53563fig3.jpg" /> 图3: 在体内 脱乙酰测定 HEK293T细胞稳定过表达或者SIRT2或SIRT2 H187Y分别与蛋白质底物（的α-烯醇酶） ​​和帽子共转染，以增加蛋白（泳道2比1）的乙酰化水平。脱乙酰使用抗Flag抗体拉下使用抗AC-κ ​​抗体的蛋白质底物和蛋白质印迹在体内检查免疫后。乙酰化在SIRT2过度表达细胞显著降低相比于SIRT2 H187Y过度表达细胞（泳道3比4）。该SIRT2介导的脱乙酰当细胞与NAM（泳道5比3）处理蛋白质底物被抑制<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53563/53563fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 反应 </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4（可选） </td><td> 5（可选） </td></tr><tr><td>纯化的乙酰化蛋白底物（10微克） </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td></tr><tr><td>缓冲液B </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td></tr><tr><td>纯化SIRT2（2微克） </td><td> - </td><td> + </td><td> + </td><td> - </td><td> + </td></tr><tr><td> NAD +（1mμ的） </td><td> - </td><td> - </td><td> + </td><td> + </td><td> +</td></tr><tr><td>纯化SIRT2 H187Y（2微克） </td><td> - </td><td> - </td><td> - </td><td> + </td><td> - </td></tr><tr><td> NAM（10毫米） </td><td> - </td><td> - </td><td> - </td><td> - </td><td> + </td></tr></tbody></table> 表1:反应的成分。 <table fo:keep-together.within-page="1"><tbody><tr><td> 样本 </td><td> 过度 </td><td> 击倒 </td></tr><tr><td> 1 </td><td> PCDH - 普罗-GFP-空载体</td><td> pLKO1空载体或Si CTR </td></tr><tr><td> 2 </td><td> PCDH - 普罗-GFP-SIRT2标志</td><td> pLKO1 SH SIRT2 1或Si SIRT2 1 </td></tr><tr><td> 3 </td><td> PCDH -普罗-GFP-SIRT2 H187Y -flag </td><td> pLKO1 SH SIRT2 2或Si SIRT2 2 </td></tr></tbody></table> 表2:蛋白质样品。

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Nanyang Technological University

Research

JoVE Journal

Chemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

8.2K 次观看.  Feinberg School of Medicine, Northwestern University. This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

视频: Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

科研

生物化学

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

癌症研究

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

使用阿基-树脂辅助捕获检测蛋白质S-囊肿

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

开发基于点击的检测方法，用于筛选 HAT1 酶活性

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

<meta charset="utf8"></head><body>用于蛋白质-配体相互作用的高通量热位移检测

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

<meta charset="utf8"></head><body>作者聚焦：通过热位移分析推进蛋白质的结构和生化研究

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

摘要

探索更多视频

此视频中的章节

核糖体组蛋白的体外泛化和二次基质测定

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

验证组蛋白乙酰转移酶抑制剂的检测

使用阿基-树脂辅助捕获检测蛋白质S-囊肿

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合