In vivo expression of peptidylarginine deiminase in Drosophila melanogaster.

Olena Mahneva,Monica G Risley,William W Ja,Ken Dawson-Scully,Sarah L Milton,Ciny John

doi:10.1371/journal.pone.0227822

Abstract

Peptidylarginine deiminase (PAD) modifies peptidylarginine and converts it to peptidylcitrulline in the presence of elevated calcium. Protein modification can lead to severe changes in protein structure and function, and aberrant PAD activity is linked to human pathologies. While PAD homologs have been discovered in vertebrates—as well as in protozoa, fungi, and bacteria—none have been identified in Drosophila melanogaster, a simple and widely used animal model for human diseases. Here, we describe the development of a human PAD overexpression model in Drosophila. We established fly lines harboring human PAD2 or PAD4 transgenes for ectopic expression under control of the GAL4/UAS system. We show that ubiquitous or nervous system expression of PAD2 or PAD4 have minimal impact on fly lifespan, fecundity, and the response to acute heat stress. Although we did not detect citrullinated proteins in fly homogenates, fly-expressed PAD4—but not PAD2—was active in vitro upon Ca2+ supplementation. The transgenic fly lines may be valuable in future efforts to develop animal models of PAD-related disorders and for investigating the biochemistry and regulation of PAD function.

Highlights

Peptidylarginine deiminase (PAD, EC 3.5.3.15) catalyzes the deimination, or citrullination, of peptidylarginine to peptidylcitrulline in the presence of elevated Ca2+ [1,2]
To generate human PAD-expressing transgenic fly lines, plasmids harboring PAD2 or PAD4 cDNA were first validated by demonstrating recombinant expression in E. coli and in vitro PAD activity
Western blots for PAD2 or PAD4 from E. coli protein extracts showed specific bands not present in non-transformed bacteria (Fig 1A). These bands were located near the expected molecular weights for the PAD2 and PAD4 fusion proteins (78 and 101 kDa, respectively)

Summary

Introduction

Peptidylarginine deiminase (PAD, EC 3.5.3.15) catalyzes the deimination, or citrullination, of peptidylarginine to peptidylcitrulline in the presence of elevated Ca2+ [1,2]. Protein citrullination plays an important role in a number of physiological processes. PAD4 acts as a corepressor to regulate expression of histone-associated genes [3,4]. There are five highly conserved PADs, PAD1-4 and PAD6 [5]. While PAD homologs have been identified in vertebrates including fish and avian species [6,7,8,9,10,11]—as well as in protozoa, fungi, and bacteria [12,13,14,15,16,17]—there are no known PAD homologs in Drosophila melanogaster.

Methods

Results

Conclusion