Peptidylarginine deiminase type 4: identification of a rheumatoid arthritis-susceptible gene

Recent studies using linkage disequilibrium and SNPs uncovered a rheumatoid arthritis (RA)-susceptible haplotype in the gene encoding peptidylarginine deiminase (PADI) type 4. This gene is one of four known PADI genes that encode enzymes to change arginine into citrulline in proteins. Post-translational modifications of proteins, including peptidyl citrullination, are related to autoimmunity, and peptidyl citrulline is a known target of one of the most RA-specific autoantibodies. Further research on PADI4, its citrullination of native peptides, subsequent breakdown of tolerance, and the role of these peptides in the development of RA, is expected to bring a better understanding of autoimmunity and arthritis, and advancements in the medical care of RA.     

Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro

Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation.     

肽基精氨酸脱亚胺酶4(PADI4)对RA的影响

类风湿关节炎(RA)是一种病因和发病机制尚不清楚的自身免疫疾病,一般认为是由多种遗传因素和环境因素共同作用的结果。遗传因素中以组织相容性白细胞抗原HLA最为重要,另外作为非HLA的肽基精氨酸脱亚胺酶4(PADI4)也参与了RA的发病。PADI4是一种翻译后修饰酶,可在钙离子存在的情况下将精氨酸残基转化为瓜氨酸残基,瓜氨酸化后的蛋白质往往改变其分子构象,从而导致其生化活性亦发生改变。在不用种族的人群中,PADI4基因多态性与RA的易感性不尽相同。PADI4在RA患者血清中含量明显升高,在机体内产生自身抗PADI4抗体,并且PADI4瓜氨酸化多种蛋白质引起机体自身的免疫反应参与RA的发生与发展。近些年来的其他研究表明PAID4也参与了肿瘤、溃疡性结肠炎、多发性硬化症的发病。尽管针对PADI4的研究已经取得了很多重大进展,但是仍然存在很多悬而未决的问题等待科研工作者进一步的研究和证实。     

The functional haplotype of peptidylarginine deiminase IV (S55G,A82V and A112G) associated with susceptibility to rheumatoid arthritis dominates apoptosis of acute T leukemia Jurkat cells

Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previously, our study showed that inducible PADI4 causes haematopoietic cell death. Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn't induce apoptosis however it promoted inducible PADI4-decreased cell viability and -enhanced apoptosis. Through in vitro and in vivo PADI enzyme activity assay, we demonstrated that PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis were compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c was released from mitochondria to cytoplasm in significant amounts. Western blotting data showed the increase in apoptosomal caspase activation during programmed cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 increasing their enzyme activity could enhance apoptosis through the mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs. The authors (H.-C. Hung and C.-Y. Lin) contributed equally to this paper.     

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2alpha

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71).     

Identification of citrullinated eukaryotic translation initiation factor 4G1 as novel autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis. We previously reported that functional variants of the gene encoding peptidylarginine deiminase type 4 were closely associated with RA. The purpose of this study was to investigate the citrullinated autoantigens recognized by serum samples from patients with RA. The human chondrocyte cDNA expression library was citrullinated by PADI4 and was immunoscreened with anti-modified citrulline antibodies and sera from patients with rheumatoid arthritis. One immunoreactive cDNA clone containing a 2480-base pair insert was isolated and sequence analysis revealed that the cDNA included a part of the eukaryotic translation initiation factor 4G1. Immunoreactivity against a recombinant citrullinated eIF4G1 fragment was observed with high specificity in 50.0% of RA patients. The levels of antibodies against citrullinated eIF4G1 were correlated with those of anti-CCP antibodies. Citrullinated eIF4G1 was identified as a candidate citrullinated autoantigen in RA patients. Citrullination of eIF4G1 may thus be involved in the pathogenesis of RA.     

Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation

DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other''s enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape.     

Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.     

Conversion of peanut trypsin-chymotrypsin inhibitor B-III to a chymotrypsin inhibitor by deimination of the P1 arginine residues in two reactive sites

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.