Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†

Abstract

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Keywords: anterior pituitary; citrullination; mammary gland; ovary; peptidylarginine deiminase; uterus.

Graphical Abstract

Figure 1

PAD enzymes remove the positively charged imine group from arginine (red) resulting in the neutrally charged, non-coded, residue citrulline (blue).

Figure 2

Conversion of a positive arginine (red) to neutral citrulline (blue) by PAD enzymes can alter intra- and inter-molecular interactions of citrullinated proteins, mediate proteolysis and immunogenicity, change the active site, and regulate chromatin structure.

Figure 3

PAD expression in female reproductive tissues.

Figure 4

PAD2 (green) is expressed in placentas from different species. Placental tissue samples were collected under approval of the Colorado State University IACUC committee (protocol number 1303). Individual sheep, horse, dog, and guinea pig placentas were obtained at gestational days 75 (sheep) and at term (horse, dog, guinea pig) then fixed in 4% paraformaldehyde and paraffin embedded. Five micron sections were processed for H&E staining or routine immunofluorescence using an anti-PAD2 antibody (1:100 ProteinTech, 12110-1-AP) and imaged using Olympus BX63 fluorescence microscope. Blue nuclear staining is DAPI, small inserts are no primary antibody negative controls, and scale bar represents 20 μm. Light blue in zonary and hemochorial is autofluorescence from red blood cells.

Figure 5

PAD2 and 4 are expressed in normal human endometrial tissue (n = 2/ out of 4 cores) and a subset of endometrial adenocarcinomas (n = 2/ out of 14 cores). Serial uterine tumor array slides were purchased from US Biomax (T094) and contain 12 cases with 24 cores and include associated pathology grade. Tumor array slides were examined by immunohistochemistry using an anti-PAD2 antibody (1:100 ProteinTech, 12110-1-AP), anti-PAD4 (1:100 Millipore Sigma, P4874) or an equal mass of non-specific IgG (data not shown). Images were taken with a Zeiss AxioVert.A1 with AxioCam MRc and scale bar represents 100 μm.

Figure 6

PAD2 expression is present in multiple subtypes of pituitary tumors. Pituitary tumor samples including subtype pathology were obtained from Institut D’Investigacions Biomediques August Pi i Sunyer Biobanc (Barcelona, Spain). Samples were de-identified and thus were exempt from University of Wyoming institutional review board (IRB) approval (protocol number 20140814BC00496). Tumors (n = 4 /tumor subtype) were examined by immunohistochemistry using an anti-PAD2 antibody (1:100 ProteinTech, 12110-1-AP) or an equal mass of non-specific IgG. Images were taken with a Zeiss AxioVert.A1 with AxioCam MRc and scale bar represents 50 or 100 μm.