Atractylenolide II Suppresses Glycolysis and Induces Apoptosis by Blocking the PADI3-ERK Signaling Pathway in Endometrial Cancer Cells.

Atractylenolide II (AT-II), the major bioactive compound of Atractylodes macrocephala, exhibits anti-cancer activity against many types of tumors, but the roles and the potential mechanisms in endometrial cancer remain unclear. In the present study, AT-II treatment was found to significantly suppress RL95-2 and AN3CA cell proliferation and glycolysis, and induced their apoptosis by inactivating the ERK signaling pathway, accompanied by the changing expression of the glycolytic key enzymes and apoptotic-related proteins. Peptidyl arginine deiminase 3 (PADI3), as the candidate target gene of AT-II, was highly expressed in the endometrial cancer tissues and associated with a poor prognosis according to bioinformatics analysis. PADI3 knockdown inhibited proliferation and glycolysis in endometrial cancer cells and induced cell apoptosis. Furthermore, AT-II negatively regulated the expression of PADI3, and PADI3 overexpression reversed the effects of AT-II on endometrial cancer cells. Our findings suggested that the anti-cancer function of AT-II is associated with the suppression of glycolysis and induction of apoptosis by blocking the PADI3-ERK signaling pathway. Thus, AT-II represents a novel therapeutic target for endometrial cancer and targeting AT-II may serve as a potential strategy for the clinical therapy of endometrial cancer.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

Structures of human peptidylarginine deiminase type III provide insights into substrate recognition and inhibitor design.

Peptidylarginine deiminase type III (PAD3) is an isozyme belonging to the PAD enzyme family that converts arginine to citrulline residue(s) within proteins. PAD3 is expressed in most differentiated keratinocytes of the epidermis and hair follicles, while S100A3, trichohyalin, and filaggrin are its principal substrates. In this study, the X-ray crystal structures of PAD3 in six states, including its complex with the PAD inhibitor Cl-amidine, were determined. This structural analysis identified a large space around Gly374 in the PAD3-Ca2+-Cl-amidine complex, which may be used to develop novel PAD3-selective inhibitors. In addition, similarities between PAD3 and PAD4 were found based on the investigation of PAD4 reactivity with S100A3 in vitro. A comparison of the structures of PAD1, PAD2, PAD3, and PAD4 implied that the flexibility of the structures around the active site may lead to different substrate selectivity among these PAD isozymes.

Citrullination of pyruvate kinase M2 by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation.

Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

Palmitoyl Acyltransferase Activity of ZDHHC13 Regulates Skin Barrier Development Partly by Controlling PADi3 and TGM1 Protein Stability.

Deficiency of the palmitoyl-acyl transferase ZDHHC13 compromises skin barrier permeability and renders mice susceptible to environmental bacterial infection and inflammatory dermatitis. It had been unclear how the lack of ZDHHC13 proteins resulted in cutaneous abnormalities. In this study, we first demonstrate that enzymatic palmitoylation activity, rather than protein scaffolding, by ZDHHC13 is essential for skin barrier integrity, showing that knock-in mice bearing an enzymatically dead DQ-to-AA ZDHHC13 mutation lost their hair after weaning cyclically, recapitulating knockout phenotypes of skin inflammation and dermatitis. To establish the ZDHHC13 substrates responsible for skin barrier development, we employed quantitative proteomic approaches to identify protein molecules whose palmitoylation is tightly controlled by ZDHHC13. We identified over 300 candidate proteins that could be classified into four biological categories: immunological disease, skin development and function, dermatological disease, and lipid metabolism. Palmitoylation of three of these candidates-loricrin, peptidyl arginine deiminase type III, and keratin fiber crosslinker transglutaminase 1-by ZDHHC13 was confirmed by biochemical assay. Palmitoylation was critical for in vivo protein stability of the latter two candidates. Our findings reveal the importance of protein palmitoylation in skin barrier development, partly by promoting envelope protein crosslinking and the filaggrin processing pathway.

Peptidylarginine Deiminase Inhibitor Cl-Amidine Attenuates Cornification and Interferes with the Regulation of Autophagy in Reconstructed Human Epidermis.

Deimination, a post-translational modification catalyzed by a family of enzymes called peptidylarginine deiminases (PADs), is the conversion of arginine into citrulline residues in a protein. Deimination has been associated with numerous physiological and pathological processes. Our aim was to study its implication in the homeostasis of human epidermis, where three PADs are expressed, namely PAD1, 2, and 3. Three-dimensional reconstructed human epidermis (RHEs) were treated for 2 days with increased concentrations (0-800 µM) of Cl-amidine, a specific PAD inhibitor. Cl-amidine treatments inhibited deimination in a dose-dependent manner and were not cytotoxic for keratinocytes. At 800 µM , Cl-amidine was shown to reduce deimination by half, alter keratinocyte differentiation, decrease the number of corneocyte layers, significantly increase the number of transitional cells, induce clustering of mitochondria and of heterogeneous vesicles in the cytoplasm of granular keratinocytes, and upregulate the expression of autophagy proteins, including LC3-II, sestrin-2, and p62/SQSTM1. LC3 and PADs were further shown to partially co-localize in the upper epidermis. These results demonstrated that Cl-amidine treatments slow down cornification and alter autophagy in the granular layer. They suggest that PAD1 and/or PAD3 play a role in the constitutive epidermal autophagy process that appears as an important step in cornification.