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

Summary

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.

Abstract

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.

Introduction

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 proteins^1-3. 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 termi....

Protocol

1. PAD4 Transformation, Expression, and Purification

1. 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.
   1. Thaw 50 μl of previously prepared chemically competent BL21(DE3) cells on ice and mix with 1 μl of the pGEX plasmid containing PAD4 gene in a 5 ml culture tube. Incubate the mixture on ice for 10 min while gently shaking every 2 min.
   2. Heat shock the cells by placing th....

Results

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 °C, the fluorescence was measured with a 340/40 – 475/15 nm filter (Figure 2A). As expected, the fluorescence levels remained low as the fluorophore within ZRcoum (or the citrullinated ZRcoum) remained in .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth, Miller (LURIA-BERTANI)	Amresco	J106-2KG	http://www.amresco-inc.com
Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents	Fisher Scientific	BP902-25	http://www.fishersci.com
Isopropylthiagalactonse (IPTG)	Life Technogolies	15529-019	http://www.lifetechnologies.com
All Buffer Salts	Fisher Scientific		Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.
Protino Glutathione (GSH) agarose	Machery-Nagel	745500.10	Store at 4 °C; http://www.mn-net.com/
Chloroamidine	Cayman Chemical	10599	Store at -20 °C; https://www.caymanchem.com/app/template/Home.vm
Triton X-100	Sigma Aldrich	X100-100ML	www.sigmaaldrich.com
Corning/Nunclon MicroWell plates (96 and 384)			Purchase from Corning; Sigma-Aldrich
Tecan Infinite 200 / Tecan i-control microplate reader software			www.tecan.com
Europium Ex. 340/40 Em. 475/15 filter			http://www.perkinelmer.com