Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Šresolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

PADI6: What we know about the elusive fifth member of the peptidyl arginine deiminase family.

Peptidyl arginine deiminase 6 (PADI6) is a maternal factor that is vital for early embryonic development. Deletion and mutations of its encoding gene in female mice or women lead to early embryonic developmental arrest, female infertility, maternal imprinting defects and hyperproliferation of the trophoblast. PADI6 is the fifth and least well-characterized member of the peptidyl arginine deiminases (PADIs), which catalyse the post-translational conversion of arginine to citrulline. It is less conserved than the other PADIs, and currently has no reported catalytic activity. While there are many suggested functions of PADI6 in the early mouse embryo, including in embryonic genome activation, cytoplasmic lattice formation, maternal mRNA and ribosome regulation, and organelle distribution, the molecular mechanisms of its function remain unknown. In this review, we discuss what is known about the function of PADI6 and highlight key outstanding questions that must be answered if we are to understand the crucial role it plays in early embryo development and female fertility. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

The PAD4 inhibitor GSK484 enhances the radiosensitivity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for approximately 10-20% of all breast cancers and is one of the leading causes of mortality among females. Radiotherapy is essential during the treatment of breast cancer. Growing evidence has indicated that peptidyl arginine deiminase-4 (PAD4) inhibitor can alleviate the development of multiple cancers, including breast cancer, through inhibiting cell proliferation. GSK484 is considered to be a highly potent PAD4-selective inhibitors. However, the potential role and mechanism of GSK484 in TNBC remain unclear. In this study, we intended to explore the effects of GSK484 on the radiosensitivity of TNBC cell lines (MDA-MB-231 and BT-549). We found that the pretreatment of GSK484 enhanced irradiation (IR)-induced inhibitory effects on cell proliferation, migration and invasion. Besides, our findings revealed that GSK484 facilitated TNBC cell apoptosis. IR treatment-induced increase of the protein level of ATG5 and ATG7, and decrease of p62 protein level were countervailed by GSK484. In addition, GSK484 enhanced DNA damage induced by IR. Moreover, in vivo experiments demonstrated that the combined treatment of IR and GSK484 showed an obvious decline of tumor growth in contrast to IR-alone or GSK484-alone treatment. Overall, GSK484 may serve as a radiosensitizer of TNBC through inhibiting IR-induced autophagy.

Biallelic PADI6 variants cause multilocus imprinting disturbances and miscarriages in the same family.

The term multilocus imprinting disturbance (MLID) describes the aberrant methylation of multiple imprinted loci in the genome, and MLID occurs in patients suffering from imprinting disorder carrying methylation defects. First data indicate that functional variants in factors expressed from both the fetal as well as the maternal genome cause MLID. Molecular changes in such genes of the maternal genome are called maternal effect variants, they affect members of the subcortical maternal complex (SCMC) in the oocyte which plays an important role during early embryonic development. Whereas the contribution of variants in the SCMC genes NLRP2, NLRP5, NLRP7, and KHDC3L to the etiology of reproductive failure and aberrant imprinting is widely accepted, the involvement of PADI6 variants in the formation of MLID is in discussion. We now report on the identification of biallelic variants in a woman suffering from different miscarriages and giving birth to two children with MLID. Thereby the role of PADI6 in maintaining the proper imprinting status during early development is confirmed. Thus, PADI6 variants do not only cause (early) pregnancy losses, but maternal effect variants in this gene cause the same spectrum of pregnancy outcomes as variants in other SCMC encoding genes, including chromosomal aberrations and disturbed imprinting. The identification of maternal effect variants requires genetic and reproductive counseling as carriers of these variants are at high risks for reproductive failure.

The subcortical maternal complex protein Nlrp4f is involved in cytoplasmic lattice formation and organelle distribution.

In mammalian oocytes and embryos, the subcortical maternal complex (SCMC) and cytoplasmic lattices (CPLs) are two closely related structures. Their detailed compositions and functions remain largely unclear. Here, we characterize Nlrp4f as a novel component associated with the SCMC and CPLs. Disruption of maternal Nlrp4f leads to decreased fecundity and delayed preimplantation development in the mouse. Lack of Nlrp4f affects organelle distribution in mouse oocytes and early embryos. Depletion of Nlrp4f disrupts CPL formation but does not affect the interactions of other SCMC proteins. Interestingly, the loss of Khdc3 or Tle6, two other SCMC proteins, also disrupts CPL formation in mouse oocytes. Thus, the absence of CPLs and aberrant distribution of organelles in the oocytes caused by disruption of the examined SCMC genes, including previously reported Zbed3, Nlrp5, Ooep and Padi6, indicate that the SCMC is required for CPL formation and organelle distribution. Consistent with the role of the SCMC in CPL formation, the SCMC forms before CPLs during mouse oogenesis. Together, our results suggest that the SCMC protein Nlrp4f is involved in CPL formation and organelle distribution in mouse oocytes.