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The maternal PADI6–UHRF1–UBE2D complex regulates ubiquitination during oocyte maturation and embryogenesis

Nature Structural & Molecular Biology volume 33, pages 512–524 (2026)Cite this article

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Abstract

Proteostasis in mammalian oocytes is vital for successful reproduction. The cytoplasmic lattices (CPLs) of oocytes store essential maternal proteins for early embryo development. Here we show that PADI6, a core component of CPLs, forms a conserved ternary complex that we term MPU for maternal PADI6–UHRF1–UBE2D. The MPU complex regulates protein ubiquitination during oocyte maturation and early embryogenesis. We determined the cryo-electron microscopy structure of MPU and show that 86% (25/29) of clinically identified PADI6 missense variants disrupt MPU assembly, revealing a potential molecular mechanism linking dysregulation of ubiquitination on oocytes to abnormal embryonic development. Mechanistically, PADI6, with the assistance of UHRF1, sequesters UBE2D to prevent ubiquitin transfer from E2 to relevant substrate proteins, thereby suppressing the ubiquitination cascade. Therefore, our findings implicate PADI6 in the regulation of proteostasis by controlling the ubiquitination cascade, expanding our understanding of PADI6-dependent regulation of oocyte maturation and early embryogenesis.

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Fig. 1: PADI6 and UHRF1 form a maternal complex.
Fig. 2: Structural insight into the assembly of hPADI6, hUHRF1 and hUBE2D.
Fig. 3: Structural mapping of the disease-related variants and their effects on human MPU formation.
Fig. 4: hPADI6 inhibits protein ubiquitination in vitro.
Fig. 5: Conserved MPU complex in mice.
Fig. 6: PADI6 deficiency results in increased ubiquitination level in mouse oocytes.
Fig. 7: MPU regulates protein ubiquitination during oocyte maturation and embryogenesis.

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Data availability

The structural coordinates and factors were deposited to the Protein Data Bank under accession code 9LPK and the cryo-EM maps were deposited to the EM Data Bank under accession code EMD-63270. The MS proteomics data were deposited to the ProteomeXchange Consortium through the iProX partner repository60,61 with the dataset identifier PXD060230. Source data are provided with this paper.

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Acknowledgements

We thank BioRender.com for providing resources used to create Figs. 1a and 7 in this publication. We thank the members (D. Du, R. Wang, X. Wu and W. Li) of the MS Platform (Frontiers Science Center for Disease-related Molecular Network, West China Hospital) for data acquisition and analysis. We thank the SKLB West China Cryo-EM Center of Sichuan University Cryo-EM support. This study was supported by the Major Program of Shenzhen Bay Laboratory (C1012513007) and the National Natural Science Foundation of China (31971132 and 32171211 to D.D.; 82001540 to Y.L.). The funders had no role in study design, data collection and analysis, publication decisions or manuscript preparation.

Author information

Author notes

  1. These authors contributed equally: Jinhong Li, Yuechao Lu, Zhili Xia.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University, Chengdu, China

    Jinhong Li, Yuechao Lu, Pengliang Chi, Qianqian Qi, Sibei Liu, Sicheng Ju, Jialu Li, Zihan Zhang, Zhuo Han, Qingting Liu, Xiang Wang, Li Guo, Wei Huang & Dong Deng

  2. Department of Reproductive Medicine, West China Second University Hospital, Sichuan University, Chengdu, China

    Yuechao Lu & Wei Huang

  3. The First Clinical Medical College, Lanzhou University, Lanzhou, China

    Zhili Xia

  4. Department of Laboratory Medicine, West China Second University Hospital, Sichuan University, Chengdu, China

    Qianqian Qi

  5. Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China

    Wenbo Meng

  6. Laboratory of Pediatric Surgery, Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu, China

    Jing Chen

  7. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Lei Li

  8. University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, China

    Lei Li

  9. National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Lunzhi Dai

  10. Frontiers Medical Center, Tianfu Jincheng Laboratory, Chengdu, China

    Lunzhi Dai & Junhong Han

  11. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China

    Junhong Han

  12. Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Shaorong Gao

  13. Development and Related Diseases of Women and Children Key Laboratory of Sichuan Province, Sichuan University, Chengdu, China

    Dong Deng

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Contributions

D.D. and S.G. supervised the project and edited the paper. Jinhong Li and Y.L. designed the study, conducted most of the experiments, analyzed the data, prepared the figures and wrote the paper. Z.X. and W.M. performed the MS experiments, analyzed the data and prepared the figures. Jinhong Li, P.C., Q.Q. and S.L. performed the construct design, protein purification, EM sample preparation, pulldown assays, co-IP, immunoblotting and data collection. Jinhong Li, P.C., Q.Q. and S.J. performed the ubiquitination activity assays, structure analysis and biochemical data analysis. Y.L. and Z.Z. collected oocytes and embryos and performed the immunofluorescence staining and immunoblotting. Jinhong Li and Jialu Li were responsible for the EM data collection, data processing and model building. Z.H. performed the IP. Q.L., J.C., X.W., L.G. and L.L. contributed to the data analysis and experiment design. W.H., L.D. and J.H. participated in the study design and supervision.

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Correspondence to Wei Huang, Lunzhi Dai, Junhong Han, Shaorong Gao or Dong Deng.

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Nature Structural & Molecular Biology thanks Andrea Riccio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 The maternal effect factor PADI6 is essential for early embryonic development in mice.

(a) Strategy for the generation of Padi6 knockout mice via the Cre-loxP system. (b) Western blotting showing PADI6 protein levels in GV oocytes from female Padi6Null, Padi6+/−, WT mice, with β-actin as the loading control. Three biologically repeated experiments were performed. (c) Immunofluorescent staining showing PADI6 protein levels in GV oocytes from female Padi6Null and WT mice. Three biologically repeated experiments were performed. Scale bar, 10 μm. (d) Fertility of female Padi6Null mice. Litter sizes of female Padi6+/− (cre, flox/+) and Padi6Null (cre, flox/flox) female mice after mating with Padi6+/+ (flox/flox), Padi6+/− (cre, flox/+) and Padi6Null (cre, flox/flox) males are presented as the means ± s.d. *Number of litters. †Number of females. (e) Representative images of WT and Padi6Null oocytes during oocyte maturation. Scale bars, 1 mm. (f) Percentages of oocytes that underwent GVBD and extruded their 1st polar body during in vitro maturation. WT (n = 122) and Padi6Null (n = 75) fully grown oocytes were collected after PMSG and HCG injection. (g) Representative images of preimplantation embryos derived from WT and Padi6Null female mice. Scale bars, 1 mm. (h) Development rates of 2-cell embryos and blastocysts after zygote collection from Padi6Null and WT mice. Number of zygotes: WT (n = 70), Padi6Null (n = 53). Rate of 2-cell/blastocyst = number of 2-cell/blastocyst/number of zygotes. In (f) and (h), n = number of oocytes analyzed, and data are presented as means ± s.d. P value were determined by unpaired, two-tailed t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., no significance. WT and Padi6Null differed significantly in blastocyst development (P < 0.0001), whereas all other comparisons were not significant. Oocyte maturation potential: GVBD, WT v.s. Padi6Null, P = 0.3744; MII, WT v.s. Padi6Null, P = 0.3627. Embryo development potential:2PN, WT v.s. Padi6Null, both groups were zygotes, P = 1.0; 2-cell, WT v.s. Padi6Null, P = 0.1977; blastocyst, WT v.s. Padi6Null, P < 0.0001.

Source data

Extended Data Fig. 2 Comparative proteomic analysis of WT and Padi6Null oocytes.

(a) Venn diagram illustrating the overlap and unique proteins identified in WT and Padi6Null oocytes. (b) Principal component analysis (PCA) of proteomic profiles demonstrates clear clustering of WT (blue) and Padi6Null (red) oocytes. (c) Bar plot illustrating the number of significantly upregulated (blue) and downregulated (red) proteins in Padi6Null oocytes compared to WT. Differential expression was assessed using an unpaired Student’s t-test (two-sided). Proteins with log2 (fold change) > 0.5 or log2 (fold change) < −0.5 and P < 0.05 were considered significantly upregulated or downregulated, respectively. (d) Volcano plot illustrating the differentially expressed proteins in Padi6Null compared to WT oocytes. Differential expression was assessed using an unpaired Student’s t-test (two-sided); proteins meeting log2 (fold change) > 0.5 or log2 (fold change) < −0.5 with P < 0.05 were highlighted as significantly upregulated (blue) or downregulated (red), respectively. Key proteins are labeled as indicated. (e-g) Venn diagrams showing the overlap of quantified proteins (e), downregulated proteins (f), and upregulated proteins (g) in Padi6Null oocytes between this study (MII stage) and Jentoft et al. (GV stage). (h) Scatter plot comparing fold changes of differentially expressed proteins from the MII stage in the GV stage. Proteins are categorized as showing consistent or inconsistent trends, and as significant or insignificant in the GV stage. (i, j) GO molecular function enrichment analysis of downregulated (i) and upregulated (j) proteins in Padi6Null oocytes at the MII stage (this study, red) and GV stage (Jentoft et al., blue). GO enrichment was performed using clusterProfiler::enrichGO (hypergeometric test), with P < 0.05 as the significance threshold. No adjustments for multiple comparisons were applied. (k) Heatmap showing the expression of shared downregulated proteins between MII- and GV-stage Padi6Null oocytes. (l) GO molecular function enrichment analysis of shared downregulated proteins between the MII and GV stages. GO enrichment was performed using clusterProfiler::enrichGO (hypergeometric test), with P < 0.05 as the significance threshold. No adjustments for multiple comparisons were applied.

Extended Data Fig. 3 In vitro reconstitution of human MPU from HEK293F cells.

(a) Representative SEC (Superose 6 Increase 10/300) of the human MPU complex purified from 293F cells. More than five biologically independent experiments were performed (n > 5). The column was calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa). The black box highlights the referenced protein from the bands of the MPU member, representing the relative mobility of endogenous UBE2D paralogs in HEK293F cells, as identified by mass spectrometry. The sequences of the unique peptides identified in the proteomic analysis are highlighted in red. The detailed information was described in Supplementary Table 3. (b) Protein identity matrix of UBE2D from humans and mice generated via the UniProt alignment tool. (c) In vitro reconstitution of human PADI6-UHRF1-UBE2D2. Purified Strep-hUHRF1 (SRA-RING construct) was incubated separately with His-hPADI6 and His-hUBE2D2, as well as in a combined mixture of all three proteins. The mixtures were pulled down using Strep resin. SDS-PAGE analysis of the eluates and input samples was performed to separate proteins, which were stained with Coomassie blue to assess their interactions. Three biologically repeated experiments were performed. (d) Flowchart of cryo-EM data processing for the complex of second peak contains nucleosomes. Details are described in the Methods section.

Source data

Extended Data Fig. 4 Flowchart of cryo-EM data processing for the human MPU complex.

(a) Representative micrograph of the cryo-EM data collection from 2,191 micrographs. (b) Workflow of the cryo-EM data processing. Details are described in the Methods section. (c) Representative micrograph of the 2D classification generated from the cryo-EM particles of human MPU complex. (d) Particle angular distribution for the map of the human MPU complex. (e) Gold-standard Fourier shell correlation (FSC) curve for the refined map of the human MPU complex.

Extended Data Fig. 5 Segmented map density of the human MPU.

The atomic structures of PADI6, UHRF1 and UBE2D3 were fitted into the cryo-EM map (white).

Extended Data Fig. 6 Comparisons of MPU members with their homologs.

(a) Alignment of hPADI4 (gray, PDB:1WDA) and hPADI6 (light sky blue and dodger blue, this study). In the middle of the panel, the surface representation of the hPADI4 active site cleft superimposed with a cartoon representation of hPADI6. The substrate BAG as part of the hPADI4 structure is displayed in yellow. D673 and N598 of hPADI6 are shown with potential hydrogen bonds represented by black dashed lines. The RMSD values for the N-terminal, middle, and C-terminal domains of PADI are 1.0 Å, 1.0 Å, and 1.2 Å, respectively. (b) Alignment of hPADI6 (gray, PDB:9FMN) and hPADI6 (light sky blue and dodger blue, this study). In the middle of the panel, a detailed view of the dimer interface of hPADI6 is presented. The residues participating in interactions and hydrogen bonds are indicated (represented by dashed lines). The RMSD values for the N-terminal, middle, and C-terminal domains of PADI are 1.0 Å, 0.8 Å, and 1.1 Å, respectively. (c) The electrostatic potential mapped onto the accessible surface area of human MPU. Regions of negative potential are colored red, those of positive potential are colored blue, and neutral regions are shown in gray. (d) Alignment of hUHRF1SRA-DNA (sage green and tan, PDB: 3CLZ) and hUHRF1SRA (gray, this study). RMSD values for hUHRF1SRA is 1.0 Å.

Extended Data Fig. 7 Evaluating the impact of the disease-associated missense variants on human MPU stability.

(a) The localization of pathogenic variants is indicated in the domains of hPADI6. The variants associated with infertility in the carrier women are represented in red, and the variants associated with MLID in the offspring are represented in medium purple, respectively. (b) Co-immunoprecipitation assay and the effects on MPU assembly of pathogenic variants of PADI6. The assays were classified into four groups, labeled cluster 1 (b), cluster 2 (c), cluster 3 (c), and cluster 4 (d) based on the phenotype and amino acid sequence of the pathogenic mutation. hPADI6 (WT or mutants) and hUHRF1 (SRA-RING construct) were co-expressed in HEK293F cells. Cells were then lysed, immunoprecipitated with anti-Flag affinity gel, and immunoblotted for Flag-tagged hUHRF1 and Strep-tagged hPADI6. IP, immunoprecipitation. The panel beside (b–d) shows statistical data for the ratio of intensity changes of IP blots, which was utilized to determine the impact on the stability of human MPU. Data represent three biologically repeated experiments. Bars represent the means ± s.d., and dots indicate individual data points. P values were obtained by unpaired, two-tailed Student’s t tests and are presented at the top of each column.

Source data

Extended Data Fig. 8 hPADI6 inhibits the protein ubiquitination in vitro.

Coomassie blue staining image of histone H3 ubiquitination assay mediated by hUHRF1 in the absence or presence of hPADI6 or pathogenic variants of hPADI6 (N598S, S336R, D349G, R352C, Q589K, P632A, and R682Q). Three biologically repeated experiments were performed.

Source data

Extended Data Fig. 9 Sequence alignment of PADIs.

Sequence alignment of human PADI homologs and mouse PADI6. The residues involved in the interactions among the subunits of human MPU were denoted with blue and red triangles. The residues involved in the dimer interface of hPADI6 were denoted with black triangles. Sequence alignment was performed using multalin (http://multalin.toulouse.inra.fr) and visualized by ESPript (https://espript.ibcp.fr).

Extended Data Fig. 10 Sequence alignment of the MPU members.

(a) Sequence alignment between human UHRF1 and mouse UHRF1. (b) Sequence alignment of human and mouse UBE2D homologs. The residues involved in the interactions among the subunits of human MPU are denoted with blue and red triangles. Sequence alignment was performed using multalin (http://multalin.toulouse.inra.fr) and visualized by ESPript (https://espript.ibcp.fr).

Supplementary information

Reporting Summary (download PDF )

Supplementary Tables 1–4 (download XLSX )

Supplementary Table 1: Quantitative proteomic analysis of WT or Padi6Null oocytes. Supplementary Table 2: Proteomic analysis of PADI6-interacting proteins. Supplementary Table 3: Identification of the MPU member UBE2D by MS. Supplementary Table 4: Light microscopy reporting table.

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Li, J., Lu, Y., Xia, Z. et al. The maternal PADI6–UHRF1–UBE2D complex regulates ubiquitination during oocyte maturation and embryogenesis. Nat Struct Mol Biol 33, 512–524 (2026). https://doi.org/10.1038/s41594-026-01758-y

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