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'.

A novel homozygous mutation in the PADI6 gene causes early embryo arrest.

BACKGROUND: It has been proved that mutations in the PADI6 gene can cause early embryo arrest. This study describes a newly discovered mutation in PADI6 that expands the genetic spectrum of early embryo arrest. METHODS: Peripheral blood of a patient diagnosed with early embryo arrest was collected for whole-exome sequencing. Sanger sequencing was performed to confirm this mutation. The effects of the variant were investigated in human embryonic kidney 293T (HEK293T) cells using western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence. RESULTS: A novel homozygous mutation in PADI6 was identified in the proband. The patient carried a frameshift insertion mutation c.558dupA (p.Thr187Asnfs*48), which was located in the protein arginine deiminase middle domain. The variant destroyed PADI6 protein expression and reduced PADI6 mRNA expression in HEK293T cells. CONCLUSIONS: The newly identified mutation in PADI6 accounts for early embryo arrest. It expands the spectrum of genetic causes and phenotypes of infertility in humans. These findings also provide an additional possible diagnostic marker for patients with recurrent in vitro fertilization/intracytoplasmic sperm injection failure.

Some infertile patients experience multiple in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure owing to recurrent early embryo arrest. However, the underlying mechanisms remain largely unknown. Due to the development of whole-exome sequencing, early embryo arrest has been confirmed as a type of Mendelian disease. This study aimed to identify the genetic cause of early embryo arrest in patients and to expand the genetic spectrum. Furthermore, it can help doctors offer better suggestions to such patients and prevent patients from suffering from multiple IVF/ICSI failures.

Novel biallelic mutations in PADI6 in patients with early embryonic arrest.

Peptidyl arginine deiminase, type VI (PADI6) is a member of the subcortical maternal complex (SCMC), which plays vital roles in mammalian embryogenesis. Most mutations in SCMC members have been reported to cause human embryonic arrest, and a total of 15 mutations in PADI6 have been shown to be responsible for early embryonic arrest according to previous studies. However, the genetic factors behind this phenotype remain to be understood in further detail. Here, we identified 13 novel mutations and 4 previously reported mutations of PADI6 in 14 patients who were diagnosed with abnormal embryonic development caused by early arrest, embryonic fragmentation, and recurrent implantation failure. Most of the mutations were predicted by in silico analysis to be deleterious or damaging to the function of PADI6. In addition, the total and East Asian population frequencies of the mutations were low or absent in the gnomAD database. Our study expands the mutational spectrum in PADI6 and will provide precise targets for genetic counseling in the future.

Genetic Variation in PADI6-PADI4 on 1p36.13 Is Associated with Common Forms of Human Generalized Epilepsy.

We performed a genome-wide association study (GWAS) to identify genetic variation associated with common forms of idiopathic generalized epilepsy (GE) and focal epilepsy (FE). Using a cohort of 2220 patients and 14,448 controls, we searched for single nucleotide polymorphisms (SNPs) associated with GE, FE and both forms combined. We did not find any SNPs that reached genome-wide statistical significance (p ≤ 5 × 10-8) when comparing all cases to all controls, and few SNPs of interest comparing FE cases to controls. However, we document multiple linked SNPs in the PADI6-PADI4 genes that reach genome-wide significance and are associated with disease when comparing GE cases alone to controls. PADI genes encode enzymes that deiminate arginine to citrulline in molecular pathways related to epigenetic regulation of histones and autoantibody formation. Although epilepsy genetics and treatment are focused strongly on ion channel and neurotransmitter mechanisms, these results suggest that epigenetic control of gene expression and the formation of autoantibodies may also play roles in epileptogenesis.

Two novel mutations in PADI6 and TLE6 genes cause female infertility due to arrest in embryonic development.

PURPOSE: This study aims to identify genetic causes of female infertility associated with recurrent failure of assisted reproductive technology (ART) characterized by embryonic developmental arrest. METHODS: We recruited infertile patients from two consanguineous families from the Reproductive Medicine Center of Guizhou Provincial People's Hospital. Peripheral blood was collected for genomic DNA extraction. Two affected individuals and their family members were performed with whole-exome sequencing and Sanger validation in order to identify possible causative genes. For further analyzing the effect of splicing mutation on mRNA integrity in vivo, TLE6 cDNA from the peripheral blood lymphocyte of the affected individual was sequenced. In addition, the possible impact of the pathogenic mutation on the structure and function of the protein were also assessed. RESULTS: Two novel homozygous mutations in the peptidylarginine deiminase type VI (PADI6) and the transducin-like enhancer of split 6 (TLE6) genes were identified in the two families. One patient carried the frameshift deletion mutation c.831_832del:p.S278Pfs*59 of the PADI6 gene and the other patient carried the splicing mutation c.1245-2 A>G of the TLE6 gene. The analysis of the mRNA from the proband's peripheral blood leukocytes confirmed aberrant splicing. CONCLUSIONS: Our findings expand the mutational spectrum of PADI6 and TLE6 associated with embryonic developmental arrest and deepen our understanding of the genetic causes of infertility with recurrent ART failure.

Novel pathogenic variants in NLRP7, NLRP5, and PADI6 in patients with recurrent hydatidiform moles and reproductive failure.

Recurrent hydatidiform moles (RHMs) are human pregnancies with abnormal embryonic development and hyperproliferating trophoblast. Biallelic mutations in NLRP7 and KHDC3L, members of the subcortical maternal complex (SCMC), explain the etiology of RHMs in only 60% of patients. Here we report the identification of seven functional variants in a recessive state in three SCMC members, five in NLRP7, one in NLRP5, and one in PADI6. In NLRP5, we report the first patient with RHMs and biallelic mutations. In PADI6, the patient had four molar pregnancies, two of which had fetuses with various abnormalities including placental mesenchymal dysplasia and intra-uterine growth restriction, which are features of Beckwith-Wiedemann syndrome and Silver Russell syndrome, respectively. Our findings corroborate recent studies and highlight the common oocyte origin of all these conditions and the continuous spectrum of abnormalities associated with deficiencies in the SCMC genes.

Expanding the genetic and phenotypic spectrum of the subcortical maternal complex genes in recurrent preimplantation embryonic arrest.

The subcortical maternal complex (SCMC) is an oocyte-to-embryo-specific maternal functional module. Some variants of SCMC genes that contribute to preimplantation embryonic arrest have been identified. However, more novel variants should be identified to broaden the genetic and phenotypic spectrum of SCMC genes and establish their roles in embryonic development. We identified 13 novel variants in the SCMC genes, TLE6, NLRP5, NLRP2, and PADI6, from 10 of a total of 50 infertile females with recurrent preimplantation embryonic arrest. Six variants in TLE6 were found in five patients with embryonic arrest, accompanied by direct cleavage and severe fragmentation at the cleavage stage. Three patients carried NLRP5 variants, and one patient each who carried NLRP2 and PADI6 variants had subsequent poor or failed fertilization and cleavage arrest with a relatively lower ratio of severely fragmented embryos. Our findings expand the genetic and phenotypic spectrum of SCMC genes associated with human embryogenesis and might help lay the foundation for the genetic diagnosis of female infertility.

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 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.

Loss-of-function maternal-effect mutations of PADI6 are associated with familial and sporadic Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance.

BACKGROUND: PADI6 is a component of the subcortical maternal complex, a group of proteins that is abundantly expressed in the oocyte cytoplasm, but is required for the correct development of early embryo. Maternal-effect variants of the subcortical maternal complex proteins are associated with heterogeneous diseases, including female infertility, hydatidiform mole, and imprinting disorders with multi-locus imprinting disturbance. While the involvement of PADI6 in infertility is well demonstrated, its role in imprinting disorders is less well established. RESULTS: We have identified by whole-exome sequencing analysis four cases of Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance whose mothers are carriers of PADI6 variants. In silico analysis indicates that these variants result in loss of function, and segregation analysis suggests they act as either recessive or dominant-negative maternal-effect mutations. Genome-wide methylation analysis revealed heterogeneous and extensively altered methylation profiles of imprinted loci in the patients, including two affected sisters, but not in their healthy siblings. CONCLUSION: Our results firmly establish the role of PADI6 in imprinting disorders. We report loss-of-function maternal-effect variants of PADI6 that are associated with heterogeneous multi-locus imprinting disturbances in the progeny. The rare finding of two siblings affected by Beckwith-Wiedemann syndrome suggests that in some cases, familial recurrence risk of these variants may be high. However, the heterogeneous phenotypes of the other pedigrees suggest that altered oocyte PADI6 function results in stochastic maintenance of methylation imprinting with unpredictable consequences on early embryo health.

New biallelic mutations in PADI6 cause recurrent preimplantation embryonic arrest characterized by direct cleavage.

PURPOSE: To investigate the pathogenesis of the recurrent preimplantation embryonic arrest characterized by direct cleavage. METHODS: Two affected individuals underwent time-lapse imaging to observe the cleavage behaviors in their final ICSI attempts. In addition, both patients were subjected to whole-exome sequencing. After the identification of possible causative genes, molecular modeling analyses were used to evaluate the possible effects of candidate mutations on protein secondary structure. RESULTS: All the bipronucleated (2PN) zygotes from both individuals presented multiple abnormal cleavage behaviors, particularly direct cleavage (DC) and subsequent cleavage arrest. Mutation analysis identified one new frameshift mutation c.1521dupC (p.S508Qfs*5) and two missense mutations c.A1117C and c.C1708T (p.T373P and p.R570C, respectively) of the PADI6 gene, which were in the protein-arginine deiminase (PAD) domain and highly conserved. CONCLUSION: This study expands the mutation spectrum of PADI6 and is the first to propose that the preimplantation embryonic arrest with concomitant abnormal cleavage behaviors, especially DC, maybe associated with PADI6 mutations.

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.

Biallelic PADI6 variants linking infertility, miscarriages, and hydatidiform moles.

Recurrent hydatidiform moles (RHM) are aberrant human pregnancies characterized by absence of, or abnormal, embryonic development, hydropic degeneration of chorionic villi, and hyperproliferation of the trophoblast. Biallelic mutations in two maternal-effect genes, NLRP7 and KHDC3L, underlie the causation of RHM in 60% of patients. We performed exome sequencing on a patient with six pregnancy losses, two miscarriages and four HM, and found no variants that affect the functions of the known genes. We found biallelic missense variants that affect conserved amino acids in PADI6 and segregate with the disease phenotype in the family. PADI6 is another maternal-effect gene and a member of the subcortical maternal complex that has been shown to have recessive variants that affect the gene function in four unrelated women with infertility who also experienced early embryonic arrest during preimplantation development after IVF. We demonstrated that PADI6 co-localizes with NLRP7 in human oocytes and preimplantation embryos and reviewed the morphology and genotypes of four products of conception from our patient. Our data expand the involvement of PADI6 to other forms of reproductive loss and highlight the commonality between infertility, miscarriages, and molar pregnancies, in some cases.

Novel mutations in genes encoding subcortical maternal complex proteins may cause human embryonic developmental arrest.

Successful human reproduction initiates from normal gamete formation, fertilization and early embryonic development. Abnormalities in any of these steps will lead to infertility. Many infertile patients undergo several failures of IVF and intracytoplasmic sperm injection (ICSI) cycles, and embryonic developmental arrest is a common phenotype in cases of recurrent failure of IVF/ICSI attempts. However, the genetic basis for this phenotype is poorly understood. The subcortical maternal complex (SCMC) genes play important roles during embryonic development, and using whole-exome sequencing novel biallelic mutations in the SCMC genes TLE6, PADI6 and KHDC3L were identified in four patients with embryonic developmental arrest. A mutation in TLE6 was found in a patient with cleaved embryos that arrested on day 3 and failed to form blastocysts. Two patients with embryos that arrested at the cleavage stage had mutations in PADI6, and a mutation in KHDC3L was found in a patient with embryos arrested at the morula stage. No mutations were identified in these genes in an additional 80 patients. These findings provide further evidence for the important roles of TLE6, PADI6 and KHDC3L in embryonic development. This work lays the foundation for the genetic diagnosis of patients with recurrent IVF/ICSI failure.

Maternal variants in NLRP and other maternal effect proteins are associated with multilocus imprinting disturbance in offspring.

BACKGROUND: Genomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance. METHODS: Whole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found. RESULTS: We now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss. CONCLUSION: The identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.

Role for PADI6 in securing the mRNA-MSY2 complex to the oocyte cytoplasmic lattices.

The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. Our previous studies have demonstrated that ribosomal component S6 is stored in the oocyte CPLs and peptidylarginine deiminase 6 (PADI6) is critical for CPLs formation. Additionally, we found that depletion of PADI6 reduced de novo protein synthesis prior to the maternal-to-embryonic transition, therefore causing embryos to arrest at the 2-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that rRNAs are dramatically decreased in Padi6 KO oocytes. We also show that the abundance and localization of mRNAs is affected upon PADI6 depletion, suggesting that mRNAs are very possibly associated with CPLs. Consistent with this observation, the amount of the major RNA binding protein, MSY2, that is associated with the insoluble fraction of the oocytes after Triton X-100 extraction is also markedly decreased in the Padi6 KO oocytes. Furthermore, treatment of the oocytes with RNase A followed by Triton X-100 extraction severely impairs the localization of PADI6 and MSY2 in oocytes. These results indicate that mRNAs, possibly in a complex with MSY2 and PADI6, are bound in the CPLs and may play a role in securing the mRNA-MSY2 complex to the CPLs.

Mutations in PADI6 Cause Female Infertility Characterized by Early Embryonic Arrest.

Early embryonic arrest is one of the major causes of female infertility. However, because of difficulties in phenotypic evaluation, genetic determinants of human early embryonic arrest are largely unknown. With the development of assisted reproductive technology, the phenotype of early human embryonic arrest can now be carefully evaluated. Here, we describe a consanguineous family with a recessive inheritance pattern of female infertility characterized by recurrent early embryonic arrest in cycles of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We have identified a homozygous PADI6 nonsense mutation (c.1141C>T [p.Gln381(∗)]) that is responsible for the phenotype. Mutational analysis of PADI6 in a cohort of 36 individuals whose embryos displayed developmental arrest identified two affected individuals with compound-heterozygous mutations (c.2009_2010del [p.Glu670Glyfs(∗)48] and c.633T>A [p.His211Gln]; c.1618G>A [p.Gly540Arg] and c.970C>T [p.Gln324(∗)]). Immunostaining indicated a lack of PADI6 in affected individuals' oocytes. In addition, the amount of phosphorylated RNA polymerase II and expression levels of seven genes involved in zygotic genome activation were reduced in the affected individuals' embryos. This phenotype is consistent with Padi6 knockout mice. These findings deepen our understanding of the genetic basis of human early embryonic arrest, which has been a largely ignored Mendelian phenotype. Our findings lay the foundation for uncovering other genetic causes of infertility resulting from early embryonic arrest.

Potential role for PADI-mediated histone citrullination in preimplantation development.

BACKGROUND: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. RESULTS: In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. CONCLUSIONS: Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.