This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

1. PAD4改造，表达和纯化<ol><li>对于PAD4改造，改造PAD4含GST融合到化学感受态中的pGEX质粒大肠杆菌 （BL21（DE3））细胞，使用以下过程蛋白质表达。准备化学感受（氯化钙）E.根据标准方法， 大肠杆菌 （BL21（DE3））的细胞。 <ol><li>解冻加入50μl预先制备的化学感受态大肠杆菌BL21（DE3）细胞在冰上，并用1微升含有PAD4基因中有5毫升培养管中的质粒pGEX质粒混合。在冰上孵育混合物10分钟，同时轻轻摇动，每2分钟。 </li><li>热量通过在42℃水浴中放置该混合物40秒电击细胞。立即将细胞质粒混合物回在冰上2分钟以使细胞恢复。加入1毫升无菌LB肉汤中的混合物中，并置于冰上1分钟。 </li><li>孵育震惊细胞的热量，在37℃下，以250rpm振荡1小时。移液器75微升转化细胞上的氨苄青霉素抗性琼脂平板孵育在37℃下搅拌15小时。商店板在4℃下。 </li></ol></li><li> PAD4表达。 <ol><li>挑1菌落大肠杆菌BL21（DE3）细胞中从氨苄青霉素琼脂平板上，并装在5ml的LB中含有1x氨苄青霉素。发生在孵化器和动摇O / N在37℃。 </li><li>转移到1升的无菌LB含有1x氨苄青霉素三水酸（MW403.45克/摩尔）的5ml的LB（首发）的。地方生长在37℃下振荡培养箱。监测生长的OD 600。当增长达到了OD 600 0.3的移动增长到16℃，振荡培养箱。 </li><li>一旦达到的OD 600为0.6，诱导的细胞用0.3毫isopropylthiagalactoside（IPTG，MW238.30克/摩尔）。让细胞在16℃振摇15小时。 </li><li>收获细胞，离心，在4000×g离心20分钟，在0℃下。倒出上清液并储存沉淀在-80℃下。 </li></ol></li><li> PAD4净化<ol><li>重悬含有表达PAD4在大肠杆菌BL21（DE3）细胞中的50mM NaCl中（分子量58.44克/摩尔），缓冲剂粒料300毫米的NaH 2 PO 4（分子量119.98克/摩尔），10毫咪唑（分子量68.077克/摩尔），0.1毫摩尔苯甲基磺酰氟（PMSF，MW174.94克/摩尔）和1mM二硫苏糖醇（DTT，154.25克/摩尔），pH值= 8.0。 </li><li>裂解通过超声处理15分钟，将细胞在4℃下。下面的超声处理，离心分离细胞裂解物在20,000×g离心20分钟，在0℃下。倒掉并保存上清。料与谷胱甘肽（GSH）的琼脂糖珠的上清液30分钟，在RT。 </li><li>通过重力排水上清GST珠/列。用4×25毫升的1×PBS（磷酸盐缓冲盐水，pH值= 8.0）在室温洗珠。 </li><li>洗脱PAD4用2×10毫升的洗脱缓冲液，50mM的三（羟甲基）氨基甲烷（Tris碱，分子量121.14克/摩尔），10毫摩尔还原型谷胱甘肽（GSH，MW307.32克/摩尔），pH值= 8.0。 </li><li>精矿10万兆瓦切邻PAD4FF离心管中，离心分离机，在4000×g离心20分钟，在4℃下。等分的蛋白在200微升体积的1.0 ml微量离心管并储存于-80℃。 </li></ol></li></ol>为缓冲区2.准备联合解决方案<ol><li>称取氯化钠（NaCl的，​​MW58.44克/摩尔），并准备一个2M的解决方案。混合解决方案，直到清楚。 </li><li>称取三（羟甲基）氨基甲烷（Tris碱，分子量121.14克/摩尔）和制备2M的溶液，pH = 8.0。混合解决方案，直到清楚。 </li><li>称取二水氯化钙（氯化钙2·2H 2 O，MW147.01克/摩尔），并准备了500毫米的解决方案。混合解决方案，直到清楚。 </li><li>称出三（2-羧乙基）膦（TCEP，MW250.19克/摩尔）和制备200mM的溶液。混合解决方案，直到明确并储存在-20℃。 </li><li>称出二硫苏糖醇（DTT，MW154.25克/摩尔）和制备1M溶液。混合解决方案，直到明确并储存在-20℃。 </li><li>准备的Triton X-100的0.5％的溶液。 </li><li>称出乙二胺四乙酸（EDTA，分子量292.24克/摩尔）和制备100mM的溶液。混合解决方案，直到清楚。 </li><li>称出Z-Arg- Arg- 7-酰氨基-4-甲基香豆素盐酸盐（ZRcoum，MW621.69克/摩尔）和制备的二甲亚砜（DMSO）中的10mM溶液。 </li></ol> 3. PAD4测定在37℃下<ol><li>从10mM ZRcoum股票，准备ZRcoum在水中的125微米的解决方案。这125微米ZRcoum解决方案将成为解决方案A. </li><li>制备的缓冲液62.5 mM氯化钠，62.5毫摩尔Tris，12.5毫摩尔氯化钙 ，6.25毫摩尔DTT，和5μM的PAD4（pH值= 8.0）。这将溶液B </li><li>准备62.5毫米氯化钠，62.5毫米的Tris，12.5毫米氯化钙和6.25毫摩尔DTT（pH值= 8.0）的缓冲。这将溶液C. </li><li>称取胰蛋白酶，结晶（从牛胰腺）和制备10毫克/毫升的100mM的EDTA。混合解决方案，直到清晰，保存于-20℃。这将解决方案D. </li><li>得到的96孔板，并指定数目的孔为对照（无PAD4）和测试孔（+ PAD4）。 </li><li>吸取160微升的溶液B为测试井和吸160微升溶液C为对照孔。孵育15分钟，在37℃。 </li><li>移液管加入40μl溶液A到所有孔中，并孵育在37℃下45分钟。 </li><li>移取10微升的dh 2 O的一半控制井的试验井的一半。向对照孔和测试控制，移液管加入10μl溶液D的孵育10分钟，在37℃的另一半。 </li><li>获得荧光读数上的多峰阅读器的345纳米的激发波长和465nm的发射波长。 </li></ol> 4. PAD4试验在室温<ol><li>从10mM ZRcoum股票，准备ZRcoum在水中的50μM的溶液。这50微米ZRcoum解决方案将成为解决方案A. </li><li>准备的100毫米钠缓冲液氯，100毫摩尔Tris，20mM的氯化钙 ，2mM的TCEP，0.02％的Triton X-100，和8微米的PAD4（pH值= 8.0）。这将溶液B </li><li>备的100 mM氯化钠，100毫摩尔Tris，20mM的氯化钙 ，2mM的TCEP和0.02％的Triton X-100（pH值= 8.0）的缓冲液。这将溶液C. </li><li>称取胰蛋白酶，结晶（从牛胰腺）和制备10毫克/毫升的100mM的EDTA。混合解决方案，直到明确并储存在-20℃。这将解决方案D. </li><li>得到384孔板和指定井对照（无PAD4）和测试孔（+ PAD4）。 </li><li>吸取25微升的溶液B为测试井和吸管25微升溶液C为对照孔。允许缓冲带/不带PAD4在孔温育20分钟，在室温。 </li><li>移液管加入25μl溶液C的向所有孔中并孵育在室温下45分钟。 </li><li>移取10微升的dh 2 O的一半控制井的试验井的一半。向对照孔和测试控制的另一半，pipettE-10微升溶液D的孵育在室温下进行10分钟。 </li><li>获得荧光读数上的多峰阅读器的345纳米的激发波长和465nm的发射波长。 </li></ol> 5. PAD4时间扫描测定法，在37℃和室温<ol><li>从10mM ZRcoum股票，准备ZRcoum在水中的125微米的解决方案。这125微米ZRcoum解决方案将成为解决方案A. </li><li>制备的缓冲液62.5 mM氯化钠，62.5毫摩尔Tris，12.5毫摩尔氯化钙 ，6.25毫摩尔DTT，和5μM的PAD4（pH值= 8.0）。这将溶液B </li><li>称取胰蛋白酶，结晶（从牛胰腺）和制备10毫克/毫升的100mM的EDTA。混合解决方案，直到明确并储存在-20℃。这将溶液C. </li><li>获得2 96孔板和吸管160微升溶液B的进＃量孔（＃井将取决于多少时间点）。 </li><li>孵育1 96孔板中，在37℃。孵育另一96孔板中于室温。 </li><li>果仁ETTE 40微升溶液A的成在两个96孔板的所有孔中，并立即吸取10微升溶液C的进t = 0的最小的孔。继续加入10μl的溶液C中的特定时间点，直到所有的孔都被淬灭。 </li><li>获得荧光读数上的多峰阅读器的345纳米的激发波长和465nm的发射波长。 </li></ol> 6. PAD4抑制试验在室温<ol><li>从10mM ZRcoum股票，准备ZRcoum在水中的83微米的解决方案。这83微米ZRcoum解决方案将成为解决方案A. </li><li>称取CL-脒（MW424.8克/摩尔），并准备了100微米的原液。这将溶液B </li><li>制备的缓冲液中的100mM的NaCl，100毫摩尔Tris，20mM的氯化钙 ，2mM的TCEP，0.02％的Triton X-100，和8微米的PAD4（pH值= 8.0）。这将溶液C. </li><li>备的100 mM氯化钠，100毫摩尔Tris，20mM的氯化钙 ，2mM的TCEP和0.02％的Triton X-100（pH值= 8.0）的缓冲液。日是将液D. </li><li>称取胰蛋白酶，结晶（从牛胰腺）和制备10毫克/毫升的100mM的EDTA。混合解决方案，直到明确并储存在-20℃。这将解决方案E. </li><li>得到384孔板和指定井对照（无PAD4），测试孔（+ PAD4），和抑制井（PAD4 +氯脒）。 </li><li>吸取25μL溶液C到测试井和吸管25微升D液为对照孔。允许缓冲带/不带PAD4在孔温育20分钟，在室温。 </li><li>移液管加入10μl溶液B的成抑制孔，孵育20分钟，在RTE。 </li><li>吸取15微升溶液A到所有孔，并培育在室温下45分钟。 </li><li>移取10微升溶液E的所有井。在室温下孵育10分钟。 </li><li>获得荧光读数上的多峰阅读器的345纳米的激发波长和465nm的发射波长。 </li></ol> 7.粗细胞裂解液测定在室温<ol><li>后从PAD4 1L的增长，在10ml的50mM Tris，50 mM氯化钠，0.1 mM的苯基甲基磺酰氟（PMSF，MW174.94克/摩尔）和1mM二硫苏糖醇（DTT，154.25克/摩尔），pH值收获的细胞重悬细胞= 8.0。 </li><li>裂解通过超声处理15分钟，将细胞在4℃下。离心细胞裂解物在20,000×g离心20分钟，在0℃下。使用100k的分子量截止离心管中浓缩该澄清的上清液至一半通过离心的体积以4,000rpm在4℃下。 </li><li>从10mM ZRcoum股票，准备ZRcoum在水中的250微米的解决方案。这250微米ZRcoum解决方案将成为解决方案A. </li><li>备的100 mM氯化钠，100毫摩尔Tris，20mM的氯化钙 ，和2mM TCEP（pH值= 8.0）的缓冲液。这将溶液B </li><li>称取胰蛋白酶，结晶（从牛胰腺）和制备10毫克/毫升的100mM的EDTA。混合解决方案，直到明确并储存在-20℃。这将溶液C. </li><li>得到黑色384孔板和设计吃井控制（无细胞裂解液）和试验井（+细胞裂解液）。 </li><li>吸取100微升的溶液B向所有孔和吸管60微升的细胞裂解液进入试验井。孵育20分钟，在RT。 </li><li>吸取40微升溶液A到所有孔，并培育在室温下45分钟。 </li><li>移取10微升溶液C到所有的孔。在室温下孵育10分钟。 </li><li>获得荧光读数上多峰读者的345纳米的激发波长和465nm的发射波长。 </li></ol>

<ol>
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Rev. 12, 318-322 (2012).</li><li>Chang, X., Fang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PADI4+and+tumourigenesis.">PADI4 and tumourigenesis.</a> Cancer Cell Int. 10, 7 (2010).</li><li>Wang, L., Chang, X., Yuan, G., Zhao, Y., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+peptidylarginine+deiminase+type+4+in+ovarian+tumors.">Expression of peptidylarginine deiminase type 4 in ovarian tumors.</a> Int. J. Biol. Sci. 6, 454-464 (2010).</li><li>Chang, X., 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=Investigating+the+pathogenic+role+of+PADI4+in+oesophageal+cancer.">Investigating the pathogenic role of PADI4 in oesophageal cancer.</a> Int. J. Biol. Sci. 7, 769-781 (2011).</li><li>Zhang, X., 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=Genome-wide+analysis+reveals+PADI4+cooperates+with+Elk-1+to+activate+c-Fos+expression+in+breast+cancer+cells.">Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.</a> PLoS Genet. 7, (2011).</li><li>Tanikawa, 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=Regulation+of+histone+modification+and+chromatin+structure+by+the+p53-PADI4+pathway.">Regulation of histone modification and chromatin structure by the p53-PADI4 pathway.</a> Nat. Commun. 3, 676 (2012).</li><li>Cui, X., 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=The+induction+of+microRNA-16+in+colon+cancer+cells+by+protein+arginine+deiminase+inhibition+causes+a+p53-dependent+cell+cycle+arrest.">The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest.</a> PLos One. 8, (2013).</li><li>Jones, J. E., Causey, C. P., Knuckley, B., Slack-Noyes, J. L., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4+(PAD4):+Current+understanding+and+future+therapeutic+potential.">Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential.</a> Curr. Opin. Drug Discov. Devel. 12, 616-627 (2009).</li><li>Li, P., 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=Regulation+of+p53+target+gene+expression+by+peptidylarginine+deiminase+4.">Regulation of p53 target gene expression by peptidylarginine deiminase 4.</a> Mol. Cell Biol. 28, 4745-4758 (2008).</li><li>Wang, 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=Anticancer+peptidylarginine+deiminase+(PAD)+inhibitors+regulate+the+autophagy+flux+and+the+mammalian+target+of+rapamycin+complex+1+activity.">Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.</a> J. Biol. Chem. 287, 25941-25953 (2012).</li><li>Slack, J. L., Causey, C. P., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4:+a+target+for+an+epigenetic+cancer+therapy.">Protein arginine deiminase 4: a target for an epigenetic cancer therapy.</a> Cell. Mol. Life Sci. 68, 709-720 (2011).</li><li>Luo, Y., Knuckley, B., Lee, Y. H., Stallcup, M. R., Thompson, P. 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+fluoroacetamidine-based+inactivator+of+protein+arginine+deiminase+4:+design,+synthesis,+and+in+vitro+and+in+vivo+evaluation.">A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation.</a> J. Am. Chem. Soc. 128, 1092-1093 (2006).</li><li>Knuckley, B., 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+specificity+and+kinetic+studies+of+PADs.">Substrate specificity and kinetic studies of PADs.</a> Biochemistry. 1, 4852-4863 (2010).</li><li>Knipp, M., Vasak, 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+colorimetric+96-well+microtiter+plate+assay+for+the+determination+of+enzymatically+formed+citrulline.">A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline.</a> Anal. Biochem. 286, 257-264 (2000).</li><li>Knuckley, B., 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+fluopol-ABPP+HTS+assay+to+identify+PAD+inhibitors.">A fluopol-ABPP HTS assay to identify PAD inhibitors.</a> Chem. Commun. 46, 7175-7177 (2010).</li><li>Bicker, K. L., Subramanian, V., Chumanevich, A. A., Hofseth, L. J., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+citrulline:+development+of+a+phenylglyoxal-based+probe+to+visualize+protein+citrullination.">Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.</a> J. Am. Chem. Soc. 134, 17015-17018 (2012).</li><li>Wang, Q., Priestman, M. A., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+protein+arginine+deiminase+activity+by+using+fluorescence+quenching:+multicolor+visualization+of+citrullination.">Monitoring of protein arginine deiminase activity by using fluorescence quenching: multicolor visualization of citrullination.</a> Angew. Chem. Int. Ed. Engl. 52, 2323-2325 (2013).</li><li>Jones, J. E., 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=Synthesis+and+screening+of+a+haloacetamidine+containing+library+to+identify+PAD4+selective+inhibitors.">Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors.</a> ACS Chem. Biol. 7, 160-165 (2012).</li><li>Dreyton, C. 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=.">.</a> Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. , (2010).</li><li>Shimoyama, 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=Deimination+stabilizes+histone+H2A/H2B+dimers+as+revealed+by+electrospray+ionization+mass+spectrometry.">Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry.</a> J. Mass Spectrom. 45, 900-908 (2010).</li><li>Luo, 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=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45, 11727-11736 (2006).</li></ol>

The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

大量的哺乳动物蛋白质都很大程度上受到酶的下列蛋白质的核糖体的生物合成中的作用进行修改。这些翻译后修饰（翻译后修饰），可以通过改变蛋白1-3的大小，电荷，结构，和低聚状态（除其他特征），大大提高了蛋白质的功能多样性。其结果是，在蛋白质结构中的改变可以导致生理后果，如蛋白降解，细胞分化，信号传导，调节基因表达，和蛋白质 - 蛋白质相互作用。而这些修改都是在所有人类蛋白质所占的比例很大普及，终端结束对组蛋白进行非常多的共价修饰4。组蛋白是一族结构蛋白，以促进基因组DNA的缩合的。非结构化组蛋白尾巴的共价修饰都进行了规范，并通过意甲酶可以催化残基（刻录机）的共价修饰，反向相同的修改（橡皮擦），以及其中的变化区分号被印记到组蛋白尾部（读卡器）5-7。事实上，大多数已知的翻译后修饰可以在组蛋白包括甲基化，磷酸化，乙酰化，SUMO化，泛素化，和瓜氨酸化8的这个短段内被观察到。 瓜氨酸化涉及肽基精氨酸转化成非吨的RNA编码的肽基-瓜氨酸（ 图1A）。的蛋白质，负责该侧链中和，是PAD的成员（对eptide 一个rginineðeiminase）蛋白家族，所有这些都是钙依赖性酶。9,10。到今天为止，PAD家庭的五名成员已被描述（PAD1，PAD2，PAD3，PAD4和PAD6）。这个家庭的每个成员似乎针对不同的细胞蛋白质，并显示单阙组织分布概况。 PAD4是该蛋白家族已知细胞核内通过一个核定位序列11的局部的唯一成员。因此，它已被证明deiminate若干核目标，包括对组蛋白H2A的精氨酸残基3的N末端 ​​尾部精氨酸侧链（H2R3），H3（H3R2，H3R17和H3R26）和H4（H4R3）12 13。而每个PAD同功酶具有特异性和一些重要的生理功能，PAD4已经获得相当多的关注，因为它在一个数均患病和健康细胞的人性化过程的作用。最近，PAD4被证明是多能性的转录网络14中的一员。既PAD4表达水平和活性示期间小鼠重编程和基态的多潜能状态升高。通过控制干细胞的基因的调节，PAD4可保留在细胞的重编程效率的关键作用。 PAD4也被牵连在形成中性粒细胞胞外的陷阱，对病原体结合后，使他们的系统过关。组蛋白通过PAD4的hypercitrullination诱导染色质，其作为用于病原性细菌的胞外陷阱的封装基体材料的解聚，从而抵挡细菌感染15,16。 此外，PAD4已经发现了一些人类疾病方面发挥积极作用。先前已证明，PAD4的异常表达与类风湿性关节炎的发病和严重程度相关联，阿尔茨海默氏症，帕金森氏病和多发性硬化17。事实上，抗瓜氨酸化蛋白的抗体的存在是类风湿性关节炎18的最可靠的和明确的诊断和预后标志物之 ​​一。同样的，失调的PAD4的活性最近已经在许多人类癌症中，包括卵巢癌，乳腺癌，肺癌，被观察ð食管癌19-22。 PAD4和癌症之间的联系已经显示出通过ELK1癌基因或经由p53肿瘤抑制蛋白22,23介导的和以前的工作已经表明，PAD4可能是一个新的抗癌治疗靶17,24,25。原则研究证明，PAD4通过的shRNA在结肠癌细胞株HCT116枯竭显露足以诱导细胞凋亡和细胞周期阻滞26。最近发展不可逆转的PAD4抑制剂导致肿块的小鼠27减少70％左右。很明显，PAD4抑制似乎充当靶向治疗，导致选择性杀伤癌细胞的同时保留未转化的细胞。 使用小分子来关闭PAD4的功能可能被证明是一个强大的新策略，以靶向癌细胞或以增加现有的癌症化学治疗剂28。不幸的是，一个PO10吨可逆PAD4抑制剂尚未被发现。使用氯/氟imidine手柄模仿精氨酸基板27,29,30和已被证明是实用的工具，理解健康和患病状态的细胞PAD4的作用已经开发了一些共价抑制剂。但是，这些分子抑制它们都具有类似功效的活性垫。因此，需要有一种简便的测定上PAD4的活性，报告是至关重要的。迄今为止，PAD4测定法已描述了从反应的比色读出31连结氨的释放，利用荧光标记的chloroamidine底物类似物为荧光偏振测定法32，依靠乙二醛和瓜氨酸33之间的酸-促进反应，并夫妇PAD4活性荧光脱猝灭步骤34。其中，只有共价修饰haloacetamidine策​​略已被证明是与高通量SCRE兼容效果图创作平台32,35,36。我们描述一个浅显的基于荧光检测的可靠测量PAD4的活性。该测定法中，其显示很强的信号 - 噪声比，分析速度，和测量的鲁棒性，具有以发现真正有效的和选择性PAD4抑制剂的潜力。

<table border="0" cellpadding="0" cellspacing="0" width="716">
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		<tr height="46">
			<td height="46" style="height:46px;width:192px;">Name of the Material/Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:267px;">Comments/ Description</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:192px;">LB Broth, Miller (LURIA-BERTANI)</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:135px;">J106-2KG</td>
			<td style="width:267px;"><a href="http://www.amresco-inc.com/">http://www.amresco-inc.com</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td>BP902-25&#160;</td>
			<td style="width:267px;"><a href="http://www.fishersci.com/">http://www.fishersci.com</a></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:192px;">Isopropylthiagalactonse (IPTG)</td>
			<td style="width:123px;">Life Technogolies</td>
			<td>15529-019</td>
			<td style="width:267px;"><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:192px;">All Buffer Salts&#160;</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:192px;">Protino Glutathione (GSH) agarose&#160;</td>
			<td style="width:123px;">Machery-Nagel</td>
			<td style="width:135px;">745500.10</td>
			<td style="width:267px;">Store at 4 &#176;C; http://www.mn-net.com/</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Z-&#8203;Arg-&#8203;Arg-&#8203;7-&#8203;amido-&#8203;4-&#8203;methylcoumarin hydrochloride (Zcoum)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>C5429</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:192px;">Chloroamidine&#160;</td>
			<td style="width:123px;">Cayman Chemical&#160;</td>
			<td style="width:135px;">10599</td>
			<td style="width:267px;">Store at -20 &#176;C; https://www.caymanchem.com/app/template/Home.vm</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:192px;">Tris(2-&#8203;carboxyethyl)&#8203;phosphine hydrochloride (TCEP HCl)</td>
			<td style="width:123px;">Thermo Scientifc</td>
			<td style="width:135px;">20490</td>
			<td style="width:267px;"><a href="http://www.piercenet.com/">http://www.piercenet.com</a></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:192px;">Triton X-100</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:135px;">X100-100ML</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Corning/Nunclon MicroWell plates (96 and 384)</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Purchase from Corning; Sigma-Aldrich</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Tecan Infinite 200 / Tecan i-control microplate reader software</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.tecan.com/">www.tecan.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Europium Ex. 340/40 Em. 475/15 filter</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.perkinelmer.com/">http://www.perkinelmer.com</a></td>
		</tr>
	</tbody>
</table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

最初，它被证明ZRcoum可以在96孔板形式的PAD4的酶活性的报告。孔用基板ZRcoum并在存在/不存在PAD4的。以下的45分钟，在37℃温育期间，荧光用一个四十〇分之三百四十零测定-一十五分之四百七十五纳米过滤器（ 图2A）。正如所料，荧光水平保持最低值内ZRcoum（或瓜氨酸ZRcoum）的荧光团保持在锁定状态。一旦加入过量的胰蛋白酶/ EDTA的?...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

PAD4活动通过亲荧光底物类似物基于荧光监测

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

fluorescence-based-monitoring-pad4-activity-via-pro-fluorescence

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Chemistry

9.8K 次观看.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.