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 Transformation, Expression, and Purification
<ol>
	<li>For PAD4 Transformation, transform the pGEX plasmid containing PAD4 GST fusion into chemically competent E. Coli (BL21(DE3)) cells for protein expression using the following procedure. Prepare chemically competent (calcium chloride) E. Coli (BL21(DE3)) cells according to standard protocols.
	<ol>
		<li>Thaw 50 &#956;l of previously prepared chemically competent BL21(DE3) cells on ice and mix with 1 &#956;l of the pGEX plasmid containing PA...

<ol>
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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. 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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...

A large number of mammalian proteins are heavily modified by the action of enzymes following the biosynthesis of proteins by the ribosome. These post-translational modifications (PTMs) can greatly increase the functional diversity of the proteome by changing the size, charge, structure, and oligomerization state (amongst other features) of proteins1-3. As a result, the change in protein structure can lead to physiological consequences, such as protein degradation, cellular differentiation, signaling, modulation in gene expression, and protein-protein interactions. While these modifications are prevalent in a large percentage of all human proteins, the terminal ends on histone proteins undergo an unusually high number of covalent modifications4. Histone proteins are a family of structural proteins that facilitate the condensation of genomic DNA. Covalent modifications of the unstructured histone tails are carried out and regulated by a series of enzymes that can catalyze the covalent modification of residues (writers), reverse the same modifications (erasers), and distinguish among the changes being imprinted onto the histone tails (readers)5-7. In fact, most of the known PTMs can be observed within this short segment of the histone including methylation, phosphorylation, acetylation, sumoylation, ubiquitination, and citrullination8.
Citrullination involves the conversion of peptidyl-arginine to the non-tRNA encoded peptidyl-citrulline (Figure 1A). The proteins, responsible for this side chain neutralization, are members of the PAD (peptide arginine deiminase) protein family, all of which are calcium-dependent enzymes.9,10. To date, five members of the PAD family have been described (PAD1, PAD2, PAD3, PAD4, and PAD6). Each member of this family appears to target distinct cellular proteins and also displays unique tissue distribution profiles. PAD4 is the only member of this protein family known to be localized within the nucleus via a nuclear localization sequence11. Accordingly, it has been shown to deiminate a number of nuclear targets, including arginine side chains on the N-terminal tails of histones H2A arginine residue 3 (H2R3), H3 (H3R2, H3R17, and H3R26) and H4 (H4R3)12,13. While each of the PAD isozymes have specific and critical physiological functions, PAD4 has received considerably more attention due to its role in a number of human processes in both diseased and healthy cells. Recently, PAD4 was shown to be a member of the pluripotency transcriptional network14. Both PAD4 expression levels and activity were shown to be elevated during reprogramming and ground-state pluripotent states in mice. By controlling the regulation of stem-cell genes, PAD4 may retain a pivotal role in cellular reprogramming efficiency. PAD4 has also been implicated in the formation of neutrophil extracellular traps, which upon binding of pathogens enable their system clearance. The hypercitrullination of histone proteins by PAD4 induces decondensation of the chromatin, which serves as the base material for the encapsulation of pathogenic bacteria by the extracellular trap, thus warding off bacterial infections15,16.
Additionally, PAD4 has been found to play an active role in a number of human diseases. It has been previously shown that aberrant expression of PAD4 is associated with the onset and severity of rheumatoid arthritis, Alzheimer&#8217;s, Parkinson&#8217;s disease, and multiple sclerosis17. In fact, the presence of anti-citrullinated protein antibodies is one of the most reliable and definitive diagnostic and prognostic biomarkers of rheumatoid arthritis18. Likewise, dysregulated PAD4 activity has recently been observed in a number of human cancers including ovarian, breast, lung, and esophageal cancers19-22. The link between PAD4 and cancer has been shown to be mediated through the ELK1 oncogene or via the p53 tumor suppressor protein22,23 and previous work has suggested that PAD4 could be a novel anti-cancer therapeutic target17,24,25. As a proof of principle study, the depletion of PAD4 via shRNA in the colorectal cancer cell line HCT116 was revealed to be sufficient for inducing apoptosis and cell cycle arrest26. A recently developed irreversible PAD4 inhibitor led to a seventy percent reduction in tumor mass in mice27. Quite remarkably, PAD4 inhibition appeared to act as a targeted therapy that resulted in selective killing of cancerous cells while sparing untransformed cells.
The use of small molecules to turn off the function of PAD4 may prove to be a powerful new strategy to target cancer cells or to augment existing cancer chemotherapeutics28. Unfortunately, a potent reversible PAD4 inhibitor has yet to be discovered. A number of covalent inhibitors have been developed using the chloro/fluoro imidine handle that mimics the arginine substrate27,29,30 and have proven to be practical tools in understanding the role of PAD4 in both healthy and diseased state cells. However, these molecules inhibit all of the active PADs with similar potency. Therefore, the need for a facile assay that reports on the activity of PAD4 is crucial. To date, PAD4 assays have been described that link the release of ammonia from the reaction to a colorimetric readout31, utilize a fluorescently labeled chloroamidine substrate analog for fluorescence polarization assay32, rely on the acid-assisted reaction between glyoxal and citrulline33, and couple the PAD4 activity to a fluorescence dequenching step34. Of these, only the covalent modifier haloacetamidine strategy has proven to be compatible with high-throughput screening platforms32,35,36. We describe a facile fluorescence based assay that reliably measures the activity of PAD4. The assay, which displays a strong signal-to-noise ratio, speed of analysis, and robustness of measurement, has the potential to discover a truly potent and selective PAD4 inhibitor.

<table border="0" cellpadding="0" cellspacing="0" width="716">
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			<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>
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			<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>
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		<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.

Initially, it was demonstrated that ZRcoum can report on the activity of PAD4 in a 96-well plate format. Wells were incubated with the substrate ZRcoum and in the presence/absence of PAD4. Following an incubation period of 45 min at 37 &#176;C, the fluorescence was measured with a 340/40 &#8211; 475/15 nm filter (Figure 2A). As expected, the fluorescence levels remained low as the fluorophore within ZRcoum (or the citrullinated ZRcoum) r...

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.

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

Research

JoVE Journal

Chemistry

9.7K ビュー.  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.