NLRP14 modulates the activity of E3 ubiquitin ligases during the oocyte-to-embryo transition

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: minor_revision

Abstract
NLRP14 is an essential maternal factor for mammalian embryonic development. Maternal ablation of NLRP14 in mice impairs DNA demethylation and calcium homeostasis in zygotes, causing early embryonic arrest. However, the underlying biochemical events remain largely unknown. Here, we identified two binding partners (KDM2A and UHRF1) of NLRP14 and further solved structures of NLRP14-KDM2A-SKP1 and NLRP14-UHRF1. Structural analysis revealed that NLRP14 modulates the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase and the RING-type E3 ubiquitin ligase UHRF1 through two distinct mechanisms. Mechanistically, NLRP14 competitively inhibits KDM2A-mediated SCF assembly or allosterically inhibits the activity of UHRF1 by occupying the E2 ubiquitin-conjugating enzyme (UBE2D) binding site of the ubiquitin-like (UBL) domain. Deletion of NLRP14 in mice increases ubiquitination levels in oocytes during maturation and after fertilization. Collectively, our findings identify NLRP14 as a dual regulator that restrains E3 ubiquitin ligase-driven ubiquitination by limiting SCF complex assembly and attenuating UHRF1 activity. This regulatory role is required to prevent excessive protein ubiquitination and maintain proteostasis during the oocyte-to-embryo transition, thereby supporting early embryonic development. Our study uncovers maternal regulation of proteostasis in oocytes and suggests that dysregulating proteostasis is an important factor in the pathogenesis of reproductive disorders.

Introduction
Before zygotic genome activation (ZGA), the oocyte-to-embryo transition predominantly relies on a series of maternal effectors.1,2 Multiple members of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein (NLRP) family, including NLRP2, NLRP4, NLRP5, NLRP9, and NLRP14, have been identified as maternal effectors essential for early embryonic development in mouse3-6. A large number of NLRP gene variants are associated with female reproductive disorders4,7,8. Most pathogenic variants of NLRP5 lead to embryo developmental arrest (EDA), resulting in severe female infertility9,10. Variants of NLRP2 and NLRP7 are associated with EDA, hydatidiform moles, miscarriages, and multilocus imprinting disorders (MLIDs) 2,11-14. Two compound heterozygous variants in NLRP14 case EDA, resulting in infertility in women15.

Recent studies have shown that NLRP14 is vital for early embryonic development15-17. Similar to other reproduction-related NLRPs, NLRP14 is explicitly expressed in gonads and early embryos15. The Nlrp14-deficient female mice are infertile, and their embryos exhibit EDA17. Nlrp14 knockout mice display defects in oocyte maturation, disrupted calcium homeostasis, and aberrant zygotic DNA methylation15-17. Interestingly, NLRP14 deficiency leads to a sharp decrease in the protein level of ubiquitin-like-containing PHD and RING finger domain protein 1 (UHRF1) in oocytes and results in its translocation from the cytoplasm to the nucleus in zygotes15,17. UHRF1, a nuclear protein in somatic cells, is an epigenetic factor that regulates DNA methylation and histone modifications18,19. In contrast to its nuclear distribution in somatic cells, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos20,21. Notably, maternal UHRF1 regulates the proper architecture of the cytoplasmic lattice and is essential for the function of both oocytes and preimplantation embryos22-24. Maintaining cytoplasmic enrichment of UHRF1 is critical for normal early embryo development. However, the mechanisms by which NLRP14 regulates these processes remain unclear.

Here, through comprehensive in vitro and in vivo investigations, we demonstrate that NLRP14 prevents excessive ubiquitination and preserves proper histone modification states in oocytes and early embryos by inhibiting SCF E3 ubiquitin ligase assembly with KDM2A and by blocking E2 ubiquitin-conjugating enzyme binding to UHRF1, thus coordinating the ubiquitination pathway and proteostasis of oocytes and early embryos.

Results
Identification of the binding partners of NLRP14
Proteomic data identified 265 NLRP14-interacting proteins in mouse oocytes17. To further identify binding partners of NLRP14, we employed AlphaFold 3 to predict the structures of NLRP14 in complex with each of these 265 candidates individually25 (Fig. 1a). The pTM and ipTM scores of the predicted complexes indicate the reliability of the predicted fold for individual subunit and the predicted relative positions of the subunits within the complex, respectively. Using a composite confidence score (0.8 × ipTM + 0.2 × pTM) with a threshold of 0.6, we identified three high-confidence potential NLRP14 binding partners: MTND (0.73), CUX1 (0.634), and KDM2A (0.626) (Fig. 1b-c and Supplementary Fig. 1a-f). MTND, an enzyme of the methionine salvage pathway (MTA), influences hepatoma growth26 and induces pathological cell migration27. CUX1 is a conserved transcription factor,28 involved in cell proliferation and differentiation29. KDM2A is a histone demethylase that erases specific demethylated histone H3 at lysine 36 (H3K36)30. KDM2A knockout mice exhibited embryonic lethality at E10.5-12.5, accompanied by severe growth defects 31. In the KDM2A conditional knockout (cKO) female mice, the patterns of H3K36me2/3 in germinal vesicle breakdown (GVBD)-stage oocytes are remarkably changed and oocyte development is blocked32. Interestingly, a well-known NLRP14-binding protein, a multi-domain containing E3 ubiquitin ligase UHRF1 participating in DNA methylation and histone modification in epigenetic regulation 15-18 , obtains a composite confidence score of only 0.506 with an ipTM score of 0.51and pTM score of 0.49 (Fig. 1b-c and Supplementary Fig. 1g-h).

To determine whether NLRP14 binds MTND, CUX1, KDM2A and UHRF1, we performed pull-down assays in vitro. As a result, KDM2A showed the strongest enrichment for NLRP14 among these candidates, whereas UHRF1 showed only weak binding to NLRP14 (Fig. 1d, Supplementary Fig. 1i). NLRP14, in turn, also pulled down KDM2A and UHRF1, respectively (Supplementary Fig. 1j-k). In contrast, MTND and CUX1 did not pull down NLRP14 (Fig. 1d and Supplementary Fig. 1i). KDM2B, similar to its paralog KDM2A ( Supplementary Fig. 2a,b), also interacted with murine NLRP14 ( Supplementary Fig. 2c). Interestingly, although human NLRP14 shares 55.5% sequence identity with its mouse orthologue, human NLRP14 only retained the capacity to pull down the human KDM2B, indicating a species-specific difference in complex formation between humans and mice (Supplementary Fig. 2b,2d). Consistently, the interaction between NLRP14 and UHRF1 is conserved in both mice and humans (Supplementary Fig.3a,b). Of note, UHRF1 is a multidomain protein composed of five domains: a ubiquitin-like domain (UBL), a tandem tudor domain (TTD), a plant homology domain (PHD), a SET- and RING-associated domain (SRA), and a really interesting new gene (RING) domain (Supplementary Fig. 3c). To identify which domain of UHRF1 is necessary for mediating the interaction with NLRP14, we generated five truncated versions of UHRF1 from two species and performed pull-down assays. These pull-down results suggest that the UBL domain of UHRF1 is necessary for its interaction with NLRP14 in both mice and humans (Supplementary Fig. 3d,e).

Notably, deletion of NLRP14 in mice resulted in significant changes in KDM2A and UHRF1 (Fig. 1e,f). Firstly, in comparison to the wild-type (WT), the GV oocytes of Nlrp14null mice presented increased nuclear enrichment of KDM2A (Fig. 1e, Supplementary Fig. 4a). Interestingly, KDM2A demethylates H3K36me2 through its JmAjC domain30. On account of the increased nuclear accumulation of KDM2A in GV oocytes caused by the deletion of NLRP14, we hypothesize that it will influence H3K36me2 methylation levels within the nucleus. We compared immunofluorescence (IF) staining for H3K36me2 in GV oocytes from wild-type and Nlrp14null mice. Notably, IF staining demonstrated that NLRP14 deficiency reduced H3K36me2 levels in GV oocytes (Supplementary Fig. 4b-e). Consequently, NLRP14 deficiency results in increased nuclear enrichment of KDM2A and decreased H3K36me2. On the other hand, UHRF1 is decreased in GV and Metaphase II (MⅡ) oocytes and enriched in the nuclei of zygotic and two-cell embryos upon NLRP14 deficiency (Fig. 1f), consistent with a previous report33.

To further validate the interaction between NLRP14 and KDM2A/UHRF1 in vivo, we performed IF staining, co-immunoprecipitation (coIP), and proximity ligation assays (PLA) in oocytes, zygotes, and two-cell embryos. The IF staining revealed that KDM2A colocalized with NLRP14 in GV and MⅡ oocytes, whereas UHRF1 colocalized with NLRP14 in the cytoplasm during oocyte maturation and early embryonic development (Fig. 1g,h). Furthermore, the coIP assays confirmed the interactions between NLRP14 and KDM2A/UHRF1 in vivo (Fig. 1i,j). In addition, the PLA foci signals of NLRP14-KDM2A diffused in the cytoplasm of oocytes, while the PLA foci signals of NLRP14-UHRF1 appeared in the cytoplasm of oocytes and early embryos (Fig. 1k,l). Taken together, KDM2A and UHRF1 are binding partners of NLRP14. Therefore, we selected KDM2A and UHRF1 for further structural investigation.

Molecular insights into the mouse NLRP14-KDM2A-SKP1 complex
KDM2A, also known as FBXL11 or JHDM1A, consists of an N-terminal JmjC domain (including related JmjN and Hairpin), a CxxC domain, a PHD domain, an F-box domain, and a leucine-rich repeat (LRR) domain30 (Fig. 2a). Notably, through its F-box domain, KDM2A interacts with SKP1 to participate in the formation of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex34. To determine whether NLRP14 forms a complex with KDM2A and SKP1, we performed a pull-down assay in HEK293F cells. Definitely, overexpressed NLRP14 bound to KDM2A along with SKP1 (Fig. 2b). Furthermore, the PLA assay suggested that the NLRP14-containing complex interacts with SKP1 in vivo (Fig. 2c).

To understand the molecular assembly of NLRP14-KDM2A-SKP1, we aim to determine the structure of this ternary protein complex. Based on the analysis of the interface in the predicted NLRP14-KDM2A structure ( Supplementary Fig. 5a), we reconstituted the ternary complex by coexpressing the Flag-tagged full-length NLRP14 (1-993 aa), Strep-tagged KDM2A (815-1,161 aa), and Strep-tagged full-length SKP1 (1-163 aa) in HEK293F cells. Interestingly, the size-exclusion chromatography (SEC) profile of the purified NLRP14–KDM2A–SKP1 complex showed a main peak (Peak1) and a shoulder to its left (Peak2)(Fig. 2d and Supplementary Fig. 5b), indicating that NLRP14-KDM2A-SKP1 exists in different oligomerization states. We conducted structural determinations on the samples corresponding to the two peaks. Consequently, two structures of NLRP14-KDM2A-SKP1 were solved at resolutions of 2.79 Å and 3.22 Å, respectively. (Fig. 2e, Supplementary Fig. 5-6,7a, and Supplementary Table 1). The density map of NLRP14-KDM2A-SKP1 defines the C-terminal region of NLRP14 (234-993 aa), including part of the NACHT domain and the whole LRR domain, the F-box domain and the following LRR domain of KDM2A (889-1161 aa) and SKP1 (1-163 aa) (Fig. 2a,e,f).

The NLRP14-KDM2A-SKP1 from Peak1 forms a heterotrimeric complex with a 1:1:1 stoichiometry. We further analysed the interfaces among the three subunits in detail. The LRR domain of NLRP14 interacts with the LRR domain of KDM2A, while SKP1 interacts with the F-box domain of KDM2A (Fig. 2f). The Glu639, Asp641, Ser668, Leu696, Ser725 and Asp753 of NLRP14 form hydrogen bonds with Arg1060, Gln1079, Asn1082, Val1087, Arg1092, and Arg1116 of KDM2A (Fig. 2g). The NLRP14 variants (D641A and D753A) impaired the binding with KDM2A, while KDM2A variants (R1056A, Q1079A, N1082A, and R1116A) also weakened the interaction between NLRP14 and KDM2A (Fig. 2h). SKP1 inserts a C-terminal helix (147-163 aa) into the gap between F-box and LRR of KDM2A and direct contacts with the opposite helix of F-box (Fig. 2f), similar to the interaction between SKP1 and other F-box protein in reported structure of SCF35.

Additionally, the NLRP14-KDM2A-SKP1 from Peak2 assembles into a dimer of heterotrimers (Supplementary Fig. 7a,b). Two NLRP14-KDM2A-SKP1 protomers adopt almost identical conformation as the NLRP14-KDM2A-SKP1 heterotrimer from peak1, exhibiting a root‑mean‑square deviation (RMSD) of 0.110 Å ( Supplementary Fig. 7c). Therefore, we focused on the interface between two NLRP14-KDM2A-SKP1 heterotrimers in the high oligomer complex. Two NLRP14s contact with each other and adopt an almost vertical position (Supplementary Fig. 7b). Two incomplete NACHT domains interact with each other, and the C-terminal LRR domain also interacts with the NACHT of another NLRP14 (Supplementary Fig. 7b). Interestingly, the complete NACHT domains in the predicted NLRP14-KDM2A-SKP1 complex clash with the opposite NLRP14 in the dimeric ternaries (Supplementary Fig. 7d). The proteins, interfaces, structures, and assemblies (PISA) program analysis revealed that the dimeric interface of NLRP14-KDM2A-SKP1 contains four parts: interface I and interface I’ between two NLRP14s; interface II and interface II’ between NLRP14 and another KDM2A (Supplementary Fig. 7e). The NACHT domain of NLRP14 exhibits electrostatic interactions with the LRR of NLRP14’ and KDM2A’ respectively, and vice versa. Among the two NLRP14s, K959 and E462’ further form a salt bridge. The Flag pull-down shows a significantly diminished band for NLRP14-HA in the E462A lane and a slightly diminished band for the K959A variant (Supplementary Fig. 7f). Therefore, both electrostatic and van der Waals interactions seem to play a role in complex formation. Furthermore, when the heterotrimers fraction (elution volume 14.5 mL) was re-injected into the SEC column, the peak partially shifted to the position corresponding to the heterotrimers (16 mL), indicating a dynamic equilibration feature of NLRP14-KDM2A-SKP1 in solution (Supplementary Fig. 7g,h).

NLRP14 competes with CUL1 to inhibit the formation of SCF E3 ubiquitin ligase
SCF E3 ubiquitin ligase composed of SKP1, CUL1, and an F-box protein36, mediates substrate-specific ubiquitination through variable F-box proteins (Fig. 2i). Typically, the F-box domain of KDM2A serves as a substrate adaptor for the SCFKDM2A E3 ubiquitin ligases34. This prompted us to investigate whether NLRP14 binds at the substrate-binding site of the KDM2A-SKP1 module, thereby preventing the recruitment of the scaffold subunit CUL1 and the assembly of the canonical SCF complex. Therefore, we performed a structural comparison between the NLRP14-KDM2A-SKP1 and CUL1-RBX1-SKP1-SKP2 (SCFSKP2) complexes37. Superposing the SKP1 in both structures shows an RMSD of 1.03 Å, suggesting an identical conformation of SKP1 (Fig. 2j). Meanwhile, two distinct F-box domains from KDM2A and SKP2 interact with SKP1 via the identical interface, adopting a similar position. However, the C-terminal LRR domain of KDM2A extends parallel to the CUL1 of SCFSKP2, resulting in NLRP14 steric clashes with CUL1 and suggesting NLRP14 competitively inhibits the SCF complex formation (Fig. 2k). To test this hypothesis, we employed KDM2A as bait protein and examined the impact of NLRP14 on SCFKDM2A complex formation. Our results demonstrated that the addition of NLRP14 significantly attenuates the binding between KDM2A and SCF components (CUL1, SKP1, and RBX1) (Fig. 2l). We further performed a PLA assay in GV oocytes to validate the competitive inhibitory capacity of NLRP14 in vivo. Compared to the PLA foci of SKP1-CUL1 in WT GV oocytes, Nlrp14null oocytes exhibited dramatically increasing PLA foci, suggesting an enhanced interaction between SKP1 and CUL1 (Fig. 2m,n). Therefore, NLRP14 competitively inhibits the formation of SCF E3 ubiquitin ligases in oocytes.

Structure of the human NLRP14-UHRF1 complex
To obtain the structural information of NLRP14-UHRF1, we attempted to in vitro reconstruct the mouse/human NLRP14-UHRF1 complex. Considering the disordered linker region connecting the five domains of UHRF1, we speculated that UHRF1 was highly dynamic and that obtaining the full-length UHRF1 structure would be difficult (Fig. 3a). Therefore, we coexpressed the UBL domain of UHRF1 with NLRP14 in Sf9 insect cells and obtained the human NLRP14-UHRF1 complex (Fig. 3b).

Furthermore, we evaluated the human NLRP14-UHRF1 interaction via cryo-EM (Supplementary Table 1). The cryo-EM structure of the NLRP14-UHRF1 complex was determined and refined at a resolution of 3.49 Å (Fig. 3c,d, Supplementary Fig. 8). The density map was well-defined for the UBL (1-75 aa) and NLRP14 (123-1,093 aa) (Supplementary Fig. 9). The pyrin domain (PYD) of NLRP14 was not visible, possibly due to its flexibility. The UBL adopted a classic ubiquitin fold, which is identical to the reported crystal structure of UBL (Supplementary Fig 10a). Comparing the structure of human and mouse NLRP14 revealed the absence of the nucleotide-binding domain (NBD) in the mouse ortholog, likely attributable to high flexibility in this region (Supplementary Fig. 10b,c).

Consistent with the domain-mapping results, UBL directly contacted the NBD of NLRP14 (Fig. 3d). The Arg6, Arg48, and Thr68 of the UBL formed hydrogen bonds with Glu282, Asn222, Asn265, and Glu226 in the NBD of NLRP14 (Fig. 3e). Additionally, the cation-𝜋 interaction between Phe287 of NLRP14 and Gln70 of the UBL is vital for specific recruitment (Fig. 3e). We generated four mutants of UBL (R6A, R48A, T68A, and Q70A) and three mutants of NLRP14 (E226A, F287A, and E226A/F287A) and performed pull-down assays to assess their interactions in HEK293F cells. These results revealed that four UBL variants and NLRP14 (F287A and E226A/F287A) disrupt the interaction between NLRP14 and UHRF1 (Fig. 3f).

NLRP14 inhibits the ubiquitination activity of UHRF1
The RING domain of UHRF1 possesses ubiquitin E3 ubiquitin ligase activity, enabling it to ubiquitinate itself (auto-ubiquitination) and its substrates, such as histone H338-40. Notably, the UBL domain of UHRF1 recruited UBE2 to facilitate the E3 ubiquitin ligase activity of UHRF139,40. Comprehensive investigations revealed that Arg6 and Gln70 of human UHRF1 are located at the interface between the UBL and UBE2D40. In line with the previous biochemical study40, the complex structure of the UBL-UBE2D2 predicted by AlphaFold 3 also revealed the heterodimeric interface residues Arg6 and Gln70 on UBL25 (Fig. 3g, Supplementary Fig. 11). Structural observations revealed that human NLRP14 and UBE2D2 bound to the same site on UBL, suggesting a mutually exclusive interaction between NLRP14 and UBE2D2 for UBL binding. Consistent with the reported and predicted data39,40, the pull-down assay revealed that UBE2D2 binds to the UBL (Fig. 3h). Adding purified NLRP14 significantly decreased the interaction between UBL and UBE2D2 (Fig. 3h). Collectively, we proposed that the binding of NLRP14 to UHRF1 impairs the recruitment of UBE2D, thereby inhibiting the ubiquitination activity of UHRF1 (Fig. 3i).

We further established an in vitro system to assess UHRF1's ubiquitination activity. First, we generated an N-terminal truncated UHRF1 lacking the UBL domain (residues 1-78 deletion, UHRF1 △UBL) and performed a ubiquitination assay on H3 (Fig. 3j). Consistent with previous studies39,40, the human UHRF1 showed lower activity, demonstrating the critical role of the UBL domain in UHRF1 (Fig. 3j). To further examine whether NLRP14 impacts the activity of UHRF1, we performed additional assays. Notably, adding NLRP14 reduced the ubiquitination level of H3, similar to the effect as removing the UBL domain (Fig. 3k). However, the addition of NLRP14 E226A/F287A and NLRP2 (a homolog of NLRP14) had less inhibitory effect on UHRF1 auto-ubiquitination and H3 ubiquitination (Fig. 3k). Consistently, murine NLRP14 also negatively modulate the activity of murine UHRF1, suggesting the conserved mechanism of NLRP14 in mammals (Fig. 3l). Together, these findings suggest that the binding of NLRP14 leads to decreased ubiquitination activity of UHRF1 in vitro.

NLRP14 deficiency increases protein ubiquitination levels in vivo
NLRP14 competitively inhibits the assembly of SCF E3 ubiquitin ligases and also allosterically inhibits the activity of UHRF1 via occupancy of the UBE2D binding site of the UBL domain of UHRF1 (Fig. 2 and Fig. 3). Recent studies have suggested that NLRP14 binds to UHRF1 to maintain its abundance during the early embryos development15-17. Therefore, all these observations suggest that NLRP14 plays a regulatory role in protein ubiquitination and maintains protein homeostasis in oocytes.

To confirm the role of NLRP14 in ubiquitination regulation, we examine ubiquitination in oocytes and embryos using the reported strategy (an antibody against ubiquitinated proteins, clone FK2)41. We observed significantly increased aggregation signals of ubiquitinated proteins in Nlrp14null oocytes (Fig. 4a-d). The number and average size of aggregation signals in Nlrp14null oocytes are significantly greater than those in WT oocytes (Fig. 4b,c). Consistently, a similar increase in ubiquitination was observed in mature oocytes and fertilized eggs (Fig. 4b,c).

Recent studies have shown that immature mouse oocytes sequester ubiquitinated protein aggregates within specialized super-organelles, termed endolysosomal vesicular assemblies (ELVAs), and subsequently degrade these aggregates during oocyte maturation46. ELVAs contain proteasomes and lysosomes, which are involved in two typical pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) for the degradation of ubiquitinated proteins 42,43. Thus, we used a core proteasome marker (20S proteasome) and the lysosomal marker lysosome-associated membrane glycoprotein 1 (LAMP1) to determine which ubiquitination degradation pathway was pathogenically activated by Nlrp14 knockout. Notably, the number and average size of the 20S proteasome puncta increased dramatically in Nlrp14 knockout oocytes (Fig. 4e-h). Moreover, there was a significant elevation in the average size of LAMP1 puncta in Nlrp14null oocytes (Fig. 4i-k), suggesting that the autophagy-lysosome pathway was also increased. These results indicated that NLRP14 deficiency increased protein ubiquitination during mouse oocyte maturation and fertilization. Interestingly, aggregated proteins were sequestered in ELVAs in normal oocytes41. The IF staining results revealed that LAMP1 foci in Nlrp14null oocytes were increased to those in normal oocytes, suggesting that the aggregated proteins in ELVAs could be regulated by NLRP14.

Methods
Ethical Statement
All animal care and experimental manipulations were conducted in accordance with the guidelines of the Animal Ethics Committee of West China Hospital, Sichuan University (No. 20220929004). Nlrp14-knockout mice on a C57BL/6 background were generated by Cyagen Biosciences using the CRISPR/Cas9 system as previously reported15. All mice were maintained in specific pathogen-free conditions on a 12 h light /12 h dark schedule at approximately 21-24 °C and 40-70% humidity. All mice were humanely killed via cervical dislocation. The knockout mice have been genotyped by PCR. Primers include:
KO-F1: 5’-CAGGAACACCAAAGACCATGAAG-3’
KO-R1: 5’-AAAGGACTCATACATCCAGTGGC-3’
WT-F2: 5’-GGAAACCGTGAGTGAGGACAAAG-3’
WT-R2: 5’-GTTTGTGGAGCCTCGGAAGTTTG-3’

Molecular cloning
For mammalian cell (HEK293F) expression, the human NLRP14, KDM2A (1-1162 aa, 815-1162 aa), KDM2B (942-1336 aa), UHRF1, NLRP2, and NLRP14, and murine Nlrp14, Kdm2a (815-1162 aa), Adi1, Cux1, Kdm2b (966-1312 aa), Skp1, CUL1, Rbx1 and Uhrf1 were synthesized, and the constructs were subcloned in PCAG vectors with denoted tags (Flag, Strep-tag II, HA and Flag-MBP). For insect cell (Sf9) expression, the human NLRP14 and murine Nlrp14 were cloned into a pFastBac-1 vector with Strep-tag, MBP tag, and a 3C cleavage site in tandem. The UBL domain (1-78 aa) of human and murine UHRF1 was cloned into a pFastBac-1 vector with an 8 × His-tag, MBP tag, and a 3C cleavage site in tandem. Human UHRF1 and murine Uhrf1 were cloned into the pFastBac-1 vector with a C-terminal Strep-tag II. For E.coli expression, the human UBA1 and UBE2D2 were cloned into a pET15 vector with an N-terminal 6× His tag, following a drICE cutting site. The Ubiquitin was cloned into a pET15 vector with an N-terminal 6× His tag, a drICE cutting site, and an HA tag in tandem. The murine Hist1h3a gene (histone H3) was cloned into a pET21 vector, with a C-terminal 6×His tag.

Protein expression and purification
Standard instructions were followed to generate baculovirus for protein expression in Sf9 cells (Invitrogen). For the recombination complex of human NLRP14-UHRF1, P2 viruses of Strep-MBP-hNLRP14 and His-MBP-UHRF1 were cotransfected into Sf9 cells. After 60 h of incubation, the cells were harvested by the lysis buffer (pH 8.0) containing 25 mM Tris-HCl, 150 mM NaCl, and protease inhibitors, including 0.8 µM aprotinin (Sangon Biotech, A600153), 2 µM pepstatin A (Sangon Biotech, A610583), and 5 µg/mL leupeptin (Sangon Biotech, A600580). The cells were lysed by using a high-pressure cell disruptor at 4 °C. The lysate was centrifuged at 18,000 × g for 40 min at 4 °C. The supernatant was then incubated with the Strep-Tactin resin (IBA Life Sciences, 2-4030-025). The resin was washed with lysis buffer and the tags were removed using 3C protease. The eluted protein was further purified by gel filtration (size-exclusion chromatography, SEC, Superose 6 increase, 10/300GL, GE Healthcare) in buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5 mM DTT, and the peak fractions were concentrated to 1.7 mg/mL for further cryo-EM sample preparation.

For proteins used in biochemistry assays, P2 viruses encoding His-MBP tagged mNLRP14 and Strep-tagged hUHRF1/mUHRF1 were transfected into Sf9 cells. Strep-tagged hUHRF1 (79-793 aa) or hNLRP2and hNLRP14, or hNLRP14 E226A/F287A were transfected into HEK293F cells. His-tagged Human UBA1, UBE2D2, and Ubiquitin were expressed in E.coli. Cells were collected by lysis buffer comprising protease inhibitors and lysed at 4°C using a high-pressure cell disruptor. The lysates were then centrifuged at 18,000 × g for 40 min at 4 °C. The supernatant of His-tagged proteins flowed through the Ni-NTA column (Qiagen). The resin was rinsed with lysis buffer containing 30 mM imidazole (Sigma), and the protein was eluted with lysis buffer containing 300 mM imidazole. The supernatant containing strep-tagged UHRF1 was incubated with Strep-Tactin resin. The resin was rinsed with a high-salt buffer (pH 8.0) containing 25 mM Tris-HCl and 1 M NaCl, and the protein was eluted from the resin with 50 mM biotin in lysis buffer. The supernatant of Flag-tagged proteins (hNLRP3, hNLRP14, and hNLRP14 E226A/F287A) was incubated with anti-Flag affinity gel for 1 h at 4 °C. The anti-Flag affinity gel was washed with lysis buffer. The proteins bound to the anti-Flag affinity gel were eluted by lysis buffer containing 0.2 mg/ml flag peptide. The eluted proteins were further purified by gel filtration in lysis buffer (Superose 6 Increase, 10/300 GL, GE Healthcare). Histone H3 was expressed and purified following the described protocol.51 The H3 was purified with Ni-NTA under denature buffer conditions (50 mM Tris 8.0, 500 mM NaCl, 5% glycerol, 6 M urea). H3 was refolded by dialyzing against 1 L dialysis buffer I (50 mM Tris, 500 mM NaCl, 5% glycerol, 2 M urea, 1 mM EDTA, 1 mM DTT, pH 8.0）at 4 °C for 4 h. The protein was further dialyzed against 1 L dialysis buffer II (20 mM Na3PO4, 150 mM NaCl, 3 mM KCl, 20% glycerol, 1 mM EDTA, 1 mM DTT, pH 7.0) overnight at 4 °C, and fresh cold dialysis buffer II was replaced to allow for another 4 h of dialyzation. The protein aggregate was removed by centrifugation. The purity and concentration of protein were confirmed by SDS-PAGE. All purified proteins were concentrated, aliquoted, and stored at -80 °C.

Cryo-EM sample preparation and data acquisition
Fresh human NLRP14-UHRF1 complexes were purified for electron microscopy (EM) sample preparation as described above. Quantifoil R1.2/1.3 grids (Au300, Quantifoil®) were glow-discharged at 15 mA for 60 seconds. The Vitrobot Mark IV (FEI) was used for EM sample preparation, with conditions set at 4 °C and 100% humidity. Data collection was conducted using a 300 kV Titan Krios microscope (Thermo Fisher Scientific), which was equipped with a K2 Summit detector (Gatan) and operated with EPU 2.5 software (Thermo Fisher Scientific). All movie stacks, each containing 30 frames, were collected at a nominal magnification of 130,000 ×, with a pixel size of 1.1 Å and a defocus range of -1.1 μm to -1.8 μm. Each stack was recorded for 6.0 s, with an exposure time of 0.2 s per frame. Two data collection sessions were conducted with total doses of 55.13 e-/Å² and 53.45 e-/Å², respectively.

The purified mouse NLRP14-KDM2A-SKP1 monomer/dimer complex was deposited onto glow-discharged 300-mesh alloy grids (CryoMatrix Au300-R12/13) and subsequently vitrified using the Vitrobot Mark IV. Images were acquired in counted-Nanoprobe mode on a 300 kV Titan Krios G3i electron microscope (Thermo Fisher Scientific), equipped with a Gatan K3 Summit detector and GIF Quantum energy filter. Using SerialEM software, movie stacks consisting of 50 frames each at a nominal magnification of 105,000× were collected with a pixel size of 0.85 Å and a defocus range of -1.2 μm to -1.8 μm. Each stack was recorded over 2.5 seconds, with a frame exposure of 0.05 seconds, resulting in a total electron dose of 53.63/55.22 e⁻/Å².

Single-particle data analysis
Data processing was performed using cryoSPARC version 4.1.252. A total of 4,334 movie stacks were collected and motion-corrected with 2-fold binning using MotionCor253. The the contrast transfer function (CTF) was then estimated using cryoSPARC52. Initially, 2,181 micrographs were selected. Afterward, 907,169 particles were automatically picked using a blob picker and extracted after binning (bin2) with a box size of 256 pixels. After 2D classification, the good particles were selected as templates for subsequent particle picking. A total of 1,687,452 particles were autopicked and extracted. Following 2D classification, 780,027 particles were used for ab initio reconstruction and heterogeneous refinement. A low-resolution map was yielded by using non-uniform refinement with 371,938 particles. The good particles were selected as “seed” particles. To improve the resolution of the density map, 2,143 additional movie stacks were collected. Following motion correction and CTF estimation, the particles were picked and extracted via a template picker and a blob picker. The particles were combined, and the duplicated particles were removed. Using a similar method, 894,535 particles were selected from the first dataset and combined with the particles from the second dataset. The particles were divided into six subgroups and further combined with six subgroups of the “seed” particles, respectively. A round of seed-facilitated 3D classification54 yield 715,938 high-quality particles. Several rounds of heterogeneous and homogeneous refinement were performed after the duplicated particles were removed, unbinned, and extracted, yielding a map at 3.73 Å resolution. To improve the density of the UBL domain, 3D classification was performed using a focus mask to enhance its density. After local refinement and deep EM enhancement55, we obtained a final map at 3.49 Å resolution.

For the mouse NLRP14-KDM2A-SKP1 monomer, a total of 3,807 movies were collected and processed using RELION 5.0 and CryoSPARC. Beam-induced motion correction was performed with MotionCor2 before micrographs were imported into CryoSPARC for contrast transfer function parameter estimation using PatchCTF. 6,312,912 particles were picked using Template Picker and extracted at a pixel size of 1.70 Å. Following 2D Classification, Ab-initio Reconstruction and Heterogeneous Refinement, 1,319,851 particles were screened out. These selected particles were re-extracted in RELION at a pixel size of 0.85 Å and subjected to one round of 3D classification without alignment, utilizing a mask on the complex. Ultimately, 293,321 particles underwent Non-uniform Refinement and Local Refinement after CTF refinement and particle polishing, resulting in a density map with a resolution of 2.8 Å.

For the mouse NLRP14-KDM2A-SKP1 dimer, 2,718 movies were collected and processed using RELION 5.0 and CryoSPARC. Beam-induced motion was corrected with MotionCor2, and micrographs were imported into CryoSPARC for contrast transfer function estimation using PatchCTF. A total of 4,577,153 particles were selected with Template Picker and extracted at a pixel size of 1.70 Å. After 2D Classification, Ab-initio Reconstruction, and Heterogeneous Refinement, 232,686 particles were retained. These particles were re-extracted in RELION at a pixel size of 0.85 Å and subjected to one round of 3D classification without alignment, using a mask focused on the complex. Subsequently, 49,652 particles underwent Non-uniform Refinement and Local Refinement following CTF refinement and particle polishing. Local refinements, performed with a mask on each monomer, yielded electron density maps at 3.3 Å resolution for monomer A and 3.2 Å resolution for monomer B. A composite map was generated in Chimera using the vop maximum command, followed by map sharpening using Phenix. Autosharpen to enhance structural details.

Model building and refinement
The initial structures of NLRP14-UHRF1 and NLRP14-KDM2A-SKP1 were individually generated utilizing AlphaFold 2 and AlphaFold 325,56. Subsequently, Chimera X was used to preliminarily fit the structure into the density map57. TCOOT 0.9.8.1 was then used to manually adjust the model58. The model was further refined against the cryo-EM map; this process was followed by validation through the real-space refinement tool available in PHENIX 1.20.159.

In vitro ubiquitination activity assay
The ubiquitination assay followed the described protocol with minor modifications60. Briefly, 50 nM UBA1, 1 µM UBE2D2, 5 µM Ub, 5 µM histone3 H3, and 1 µM UHRF1 or UHRF1 △UBL were incubated with or without 1 µM NLRP14, NLRP14 E226A/F287A or 1 µM NLRP2 on ice in ubiquitination assay buffer (50 mM Hepes, 50 mM NaCl, 5 mM KCl, 5 mM MgCl2, pH 7.5). The assay was started by adding 2 mM ATP and incubating at 30 °C. The ubiquitinated substrate was analysed by SDS-PAGE.

Pull-down assay, coimmunoprecipitation, and immunoblotting
For pull-down assay and coimmunoprecipitation in HEK293F cells, the constructs of NLRP14, KDM2A, KDM2B, SKP1, MTND, CUX1, CUL1, RBX1 and UHRF1 specified in the figures and figure legends were cotransfected into HEK293F cells with polyethyleneimine (25 kDa; Polysciences, 23966). The transfected cells were first cultured in a shaking incubator (37 °C, 120 rpm) for 60 h and then collected and suspended in lysis buffer. Cells were lysed by repeatedly freezing and thawing 5 times using liquid nitrogen, and the cell lysate was centrifuged at 13,000 × g for 20 min at 4 °C. The supernatant (input) was incubated with Strep-Tactin resin or anti-Flag affinity gel for 1 h at 4 °C. The Strep-Tactin resin or anti-Flag affinity gel was washed with lysis buffer containing 0.05% Triton X-100 (Sigma–Aldrich, 9036-19-5). The proteins bound to Strep-Tactin resin or anti-Flag affinity gel were eluted by lysis buffer containing 50 mM biotin or 0.4 mg/ml flag peptide. The input and eluate were analysed by immunoblotting with denoted antibodies.

For pull-down assay of UBL domain with UBE2D2 in the presence or absence of NLRP14, the purified Strep-GST-tagged UBL of hUHRF1 (30 µg) and His-Ptagged UBE2D2 (40 µg) were incubated with purified His-tagged hNLRP14 (100 µg) or an equal volume of buffer in a total volume of 400 µL system. The mixture was incubated for 1 h at 4 °C. GS4 B resin was added, and the mixture was incubated for 30 min at 4 °C. Then, the resin was washed with lysis buffer containing 0.05% Triton X-100, and the resin was eluted with lysis buffer containing 20 mM reduced glutathione (GSH) (Sigma-Aldrich). The input and eluate were analysed by immunoblotting with denoted antibodies.

The antibodies used for immunoblotting were as follows: mouse anti-Flag tag (Proteintech, 66008-4-Ig), mouse anti-Strep tag (Huaxingbio, HX1988), mouse anti-His tag (Zen-bio, 230001), mouse anti-Actin conjugated with horseradish peroxidase (HRP) (Abclonal, AC028), mouse anti-UHRF1 monoclonal antibody (Santa Cruz Biotechnology, sc-373750), rabbit anti-NLRP14 polyclonal antibody (a gift from Professor Zhonghan Li at Sichuan University), goat anti-rabbit IgG conjugated with HRP (Zen-bio, 511203), and goat anti-mouse IgG conjugated with HRP (Zen-bio, 511103).

Collection of oocytes and embryos
For oocyte and embryo collection, 6- to 9-week-old female mice were injected with 10 IU of pregnant mare serum gonadotropin (PMSG; Nanjing Aibei Biotechnology, M2630). After 46-48 h, the ovaries were scraped to release GV oocytes from the follicles in the M2 medium (Sigma-Aldrich, M7167). Mature eggs were collected at 16-18 h after an additional treatment of 5 IU human chorionic gonadotrophin (HCG, Nanjing Aibei Biotechnology, M2530). Female mice were mated with males after hCG injection, and zygotes were collected 20 hours post-mating. Two-cell embryos were obtained by culturing zygotes for 24h in a KSOM medium (Nanjing Aibei Biotechnology, M1435).

Proximity ligation assay (PLA)
The PLA was conducted with the Duolink® PLA Fluorescence kit (rabbit Plus probe DUO92002, mouse minus probe DUO92004, detection reagents DUO92014, washing buffer DUO82049) by following the manufacturer’s protocol (Sigma-Aldrich). Oocytes, zygotes, and two-cell embryos were collected and subsequently fixed with 2% paraformaldehyde (PFA, Sigma, P6148) for 60 minutes at room temperature (RT). Following fixation, the samples were permeabilized with 0.5% Triton X-100 dissolved in PBS for 30 minutes at RT and blocked with Duolink® Blocking Solution in a humidified chamber at 37 °C for 60 minutes. Primary antibodies were incubated with the samples in Duolink® Antibody Diluent at 4 °C overnight. The next day, the embryos were washed with PBSA (PBS containing 0.1% BSA). Subsequently, the samples were transferred to the probe mixture and incubated in a humidity chamber at 37 °C for 60 minutes. A washing step was then performed using 1 × Duolink® washing buffer A. Ligation of the PLA probes was carried out for 45 minutes at 37 °C, followed by amplification for 100 minutes at 37 °C in darkness. After a series of washes with 1 × Duolink® washing buffer B and 0.01 × Duolink® washing buffer B, the samples were counterstained with DAPI and visualized using an Olympus FLUOVIEW FV3000 microscope. The primary antibodies used for PLA were as follows: rabbit anti-UHRF1 polyclonal antibody (Invitrogen, PA5-29884), mouse anti-NLRP14 monoclonal antibody (Wuhan Dia-An Biotechnology Co., Ltd.), rabbit anti-KDM2A (Proteintech, 24311-1-AP), mouse anti-SKP1 (Santa Cruz, sc-5281), rabbit anti-CUL1 (Invitrogen, Catalog # MA5-54225) and mouse IgG (Beyotime, A7028)

Immunofluorescence staining and confocal microscopy
Oocytes and embryos were fixed with 2% paraformaldehyde (paraformaldehyde (Beyotime, P0099) diluted by PBS) and 0.1% TritonX 100 at room temperature (RT) for 60 minutes, washed with wash buffer (Beyotime, P0106) three times, and permeabilized with Enhanced Immunostaining Permeabilization Buffer (Beyotime, P0097) for 60 minutes at RT. The samples were blocked with blocking solution (Beyotime, P0260) at RT for 1 h and then incubated with primary antibodies overnight at 4 °C. The samples were washed in wash buffer three times and incubated with secondary antibodies conjugated fluorophore for 1 h at RT. The samples were stained with DAPI (Beyotime, C1006) or Hoechst 33258 (Beyotime, C1011) after washing with wash buffer three times. The confocal images were captured with an Olympus FLUOVIEW FV3000. The antibodies used for IF staining were as follows: mouse anti-NLRP14 monoclonal antibody (Wuhan Dia-An Biotechnology Co. LTD) and mouse anti-UHRF1 monoclonal antibody (Santa Cruz Biotechnology, sc-373750) conjugated to Alexa Fluor®647 and Alexa Fluor®555 respectively using the Apex™ Alexa Fluor™ antibody labeling kit (ThermoFisher Scientific, A20186, A20187), mouse anti-Ubiquitin monoclonal antibody (Sigma-Aldrich, ST1200), rabbit anti-LAMP1 monoclonal antibody (Cell Signaling Technology, 99437), rabbit anti-H3K36me2 (Cell Signaling Technology, C75H12) and mouse anti-20S proteasome monoclonal antibody (Santa Cruz Biotechnology, sc-58410).

Coimmunoprecipitation of oocytes and ovaries
Approximately 800 GV oocytes were harvested from 3-week-old female mice after treatment with PMSG, and these oocytes were then lysed at 4 °C for 20 minutes using the cell lysis buffer (Beyotime, #P0013) containing proteinase inhibitor cocktail (Apexbio, #K1007). Then, the protein A/G magnetic beads (Bimake, #B23201) were washed three times with the lysis buffer. The prepared beads and NLRP14 antibodies were introduced to the samples and incubated overnight at 4 °C. A total of 10 ovaries from 8-week-old female mice were collected and cut into pieces. Then these ovaries were lysed at 4 °C for 1 hour using the cell lysis buffer (Beyotime, #P0013) containing proteinase inhibitor cocktail (Apexbio, #K1007). The IgG antibody (Thermo Fisher Scientific, #02-6102) and KDM2A antibodies (Proteintech, 24311-1-AP) were introduced to the samples and incubated overnight at 4 °C. The protein A/G magnetic beads (Bimake, #B23201) were washed three times with the lysis buffer. The prepared beads were introduced to the samples and incubated for 3 hours at 4 °C. Subsequently, the proteins were precipitated and washed with IP lysis buffer (ThermoFisher Scientific, #87787). The coprecipitated proteins were eluted in 1 × SDS-PAGE loading buffer at 95 °C for 10 minutes and visualized by immunoblotting. The primary antibodies used for immunoblotting were as follows: mouse anti-NLRP14 monoclonal antibody (Wuhan Dia-An Biotechnology Co., Ltd.), mouse anti-UHRF1 monoclonal antibody (Santa Cruz Biotechnology, #sc-373750), and rabbit anti-KDM2A monoclonal antibody (Proteintech, 24311-1-AP).

Statistical analysis & Reproducibility
All experiments were performed at least three times independently with similar results, as described in the figure legends. When comparing oocytes of distinct genotypes, other experimental parameters, particularly the age of the mice, were rigorously controlled. To reduce individual differences, samples were collected from at least three mice in each experimental and control group, and at least 5 embryo/oocyte images were scanned by confocal microscopy. Fluorescence foci number and size were quantified in FIJI using the “Analyse Particle” function, selecting objects larger than 1 μm2 in the reference channel, as described by Jentoft, I.M.A. et al.46. Prism v10.6.0 was used to perform statistical analysis. A two-tailed unpaired Student’s t-test was used to compare two different groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present an interesting investigation of the maternal factor for embryonic development known as NLRP14. Following up on a prior proteomic study, the authors first validate the ubiquitin ligases UHRF1 and KDM2a as prominent interactors of NLRP14. Using both cell-based assays and structural work, the authors find that NLRP14 is found to disrupt the localization of the E3s and alter UHRF1 protein levels, and present new structures of NLRP14 bound to either KDM2a or UHR1. These structures are biochemically validated using either binding or enzyme assays. Interestingly, NLRP14 blocks KDM2a’s ability to form a stable complex with the rest of the cullin-RING complex to form an active ubiquitin ligase. For UHRF1, NLRP14 binds to UHRF1’s domain that is needed for E2 recruitment, therefore, reducing its ability to ubiquitinate its targets. The data is of good quality and well presented. Overall, the authors should consider a few relatively minor points discussed below.\n1. After the structures are presented, the authors do not circle back to examine if their structure-guided mutants would recapitulate the earlier presented phenotypes, e.g., KDM2a nuclear localization or decreased UHRF1 levels in the Nlrp14 null. These experiments will demonstrate that the effects observed are directly related to the interactions in the solved structures.\n2. In the figure 4, how are there more proteasomes or increase the size of the proteasomes? This result seems like a surprising finding unless proteasome assemblies are changing and their expression patterns are altered. Or are the authors stating that these are more aggregated proteasomes? Please expand and clarify what is happening at the molecular level. Furthermore, it is unclear if these results are directly due to the E3s discussed in the paper or the other ubiquitination machinery brought up in the discussion.\n3. E2s are not considered ubiquitin ligases. Also calling them “UBE2s” is awkward and confusing as the last letter is used to specify the E2. Please replace with Ubiquitin-conjugating enzymes or E2s or E2 enzymes. Additionally, in the results, the authors shouldn’t overstate that the Ubl mechanism is broadly applicable to all E2s that may be used by UHRF1. It is unclear if E2s other than UBE2D use the Ubl for their mechanism, assuming UHRF1 uses other E2s.\n4. The last paragraph of the abstract can be expanded to summarize their paper.\n5. The authors make this statement “A recent study on Nlrp14null mice revealed that the essential transporter NCXL on mitochondria dramatically decreased via the ubiquitylation-proteasome degradation pathway21, supporting the notion that NLRP14 inhibited the E3 activity in oocytes and embryos.“ However, unless there is evidence that is kdm2a or uhrf1 dependent, this statement seems out of place and may not be relevant.", "reviewer_2": "The presented article is based on well-conducted, state-of-the-art experiments, provides novel insights into protein degradation during oocyte maturation, which represents an actual topic. The experimental data are appropriately complemented by structural prediction of interacting proteins and many extended data. I recommend this article for publication. However, prior to publication, the following minor issues should be addressed appropriately.\nThe Discussion section should place greater emphasis on the obtained results. For example, the structure of proteins and complexes is not addressed at all, even though it constitutes a substantial portion of the Results section.\nLine 398 – 400 – It is not appropriate to describe the mentioned proteins as critical structural components of CPLs. These proteins are rather stored on CPLs. Please revise this statement accordingly.\nLine 378 – Please specify which tubulin proteins you are referring to.\nLines 123 – 125 The statement “human NLRP14 only maintains the capacity to pull down the N-terminal of human KDM2B” is confusing. Specify what is the difference between the interaction of human NLRP14 and KDM2A/B and murine NLRP14 and KDM2A/B.\nLine 315 – Please define “their” as it is not clear what it refers to.\nLine 335 – Please specify which process you are referring to.\nThe overall quality of English is good. Nevertheless, in several places it requires improvement as the inaccurate formulations reduce the quality of the paper and sometimes hinder comprehension (e.g. incorrect use of on/in changing the meaning, omission of was in the passive voice, or confusion between removement/removal and others).\nThe questions included in the Results section (lines 170 – 171 and 213 – 215) are disruptive and do not integrate well with the surrounding text. Please consider rephrasing of these sentences.\nPlease explain all abbreviation used in the manuscript, including those that are commonly known (e.g. PDB, RMSD, IF and others).\nIn the Methods section, several formulations remain written in a protocol-like style. Please revise these sentences to ensure they are appropriate in the Methods section. Line 583: are/were, Line 677/678 – whole sentence: Collect 10 ovaries from…..\nLine 697: The citation Melina et al., 2023 is not formatted as a numerical reference and is missing from the References list.\nLine 192 – typing error – analyse/analysed\nLine 346 – typing error – nuclus/nucleus", "reviewer_3": "The manuscript from the Deng lab at Sichuan University (Chengdu) describes the molecular mechanisms by which the maternal factor NLRP14 regulates early embryonic development in mammals. The authors find that NLRP14 modulates protein homeostasis during the oocyte-to-embryo transition by interacting with two types of E3 ubiquitin ligases: the SCF (SKP1–CUL1–F-box) complex and the RING-type ligase UHRF1. Using biochemical assays and cryo-EM, they show that NLRP14 inhibits SCF complex formation by competing with CUL1 for KDM2A binding. NLRP14 also blocks UHRF1’s ubiquitination activity by preventing E2 ligase recruitment to its UBL domain. In Nlrp14-deficient mice, this regulatory loss led to excessive protein ubiquitination, disrupted proteostasis, and early embryonic arrest. The findings reveal that NLRP14 safeguards protein stability during oocyte maturation and fertilization, offering new insight into maternal control of proteostasis and reproductive disorders.\nThis is a comprehensive study on the structure and function of NLRP14 in oocytes. The manuscript builds in part on a previous study from a different lab, where proteomic analysis identified 265 proteins that interact with NLRP14 in mouse oocytes (cited as reference 21). The manuscript is somewhat tedious to read, partly due to its verbose language and cumbersome explanations. This holds particularly for the structural descriptions. However, the topic of the study is interesting and timely, adding to our understanding of the oocyte-to-embryo transition, and the protein complexes analyzed are of importance.\nCriticism\nAlbeit lengthy, the description and presentation of the cryo-EM structures is not very precise and not according to the standards in structural biology. When the authors introduce recombinant formation of the NLRP14–KDM2A–SKP1 complex, the authors speak of a “noncanonical peak with a shoulder” (line 182). What is this and at which expected size is the peak eluting? The authors continue describing that they determined the structures of “NLRP14-KDM2A-SKP1 in two distinct oligomeric states” (line 186) but then describe the first one only while the second one is introduced later in line 201. It would more to the point to describe the main peak as a heterotrimer and the shoulder left of it as a dimer of this heterotrimer. How is the dimer of the ternary NLRP14–KDM2A–SKP1 complex formed? This is not well described in the paragraph starting at line 201 and also not well visible in Fig. 2g and h. I assume, it is a C2 symmetry? The authors say that the dimer formed of two trimers is “… indicating the dynamic equilibration feature of NLRP14-KDM2A-SKP1 in solution …” (line 203). Where is this shown in the Ext. Data Fig. 7a? Is this dynamic equilibrium a result of a post-translational modification or is it concentration dependent? The authors should show size exclusion chromatograms similar to Fig. 2d at varying complex concentrations in order to specify if the observed splitting in a shoulder and main peak is a concentration dependent dynamic equilibrium. If the splitting would be dependent on a PTM, that occurs for example during the expression, the authors should perform comparative peptide mass finger-print analysis, e.g. from the fractions shown in ED Fig. 5b.\nIn Extended Data Figure 7 c and d, the subunits are not labelled. As an intermolecular contact, shouldn’t, e.g., R357 be labelled as R357’ according to the standards?\nThe dimer formation of the NLRP14-KDM2A-SKP1 complex should be shown from a better perspective. Either in ED Fig. 7a, b or in Fig. 2.\nLine 205f: “… the dimeric interface of NLRP14-KDM2A-SKP1 suggests two pairs of polar contacts (E462 and K959) …” In which subunit(s)? Please specify.\nLine 207f: “Interestingly, the NLRP14 variants E462 and K959 cannot disrupt the dimer, …” Which variants? I can identify the mutants E462A and K959A marked in ED Fig. 7e.\nWhile I could continue here, the entire description of the complex structures should be substantially revised and improved.\nThe mutually exclusive binding properties between NLRP14 and UBL compared to UBE2D2 and UBL could be described a bit better and in other terms (‘mutually exclusive’, ‘displacement’). Can the authors determine the dissociation constants of these interactions?\nIt is surprising that the cryo-EM structure determination of NLRP14 in two different complexes is not mentioned in the abstract, despite two figures in the main text describing the structural analysis, as also most of the extended data figures do and the ED table. Please correct this.\nAbstract, line 47: “… thereby establishing a theoretical framework for further investigation …” What does ‘theoretical framework’ mean. Please specify.\nThe figure legends are rather lengthy, maybe they can be shortened.\nDiscussion: The paragraph starting in line 389 (“Proteostasis is critical …”) is very good and describes the rational of the study. I wonder, if this could be a good starting paragraph for the discussion section, to provide some more background to the reader. By the way, “Proteins in oocytes exhibit an exceptionally long lifespan, with half-lives significantly exceeding those found in other cell types51.” (line 391). Can the authors specify the lifespan in terms of time?\nThe model Figure 5 is not very compelling. Could the authors put this into a bigger picture, particularly with respect to the importance of protostasis in oocytes as described in the discussion section (see above). The essence of this study, that NLRP14 inhibits ubiquitination activity by interacting with two different E3 ligase complexes, should be emphasized more strongly in such a cartoon.\nMinor\nLine 56,57: “Mutations in NLRPs are associated with female reproductive diseases4,7,8.” You may change diseases to ‘disorders’ or ‘dysfunctions’.\nLine 123f: “Interestingly, human NLRP14 only maintains the capacity to pull down the N-terminal of human KDM2B, …” This sentence is not clear to me.\nLine 193: “monomeric NLRP14-KDM2A-SKP1 complex.” might be changed to ‘globular’, ‘heterotrimeric’ or ‘ternary’.\nLine 313: “Therefore, all these ‘observations’ suggest that …“\nThe degree of rotation in Fig. 3c should be specified (I assume 90°).\nSupplements, line 23: The conserved interaction …\nSupplements, line 41: The conserved interaction …\nSupplements, line 110: Residues mediating interactions between XXX and XXX are shown. I am unable to align the displays in panels c and d with the boxes in panel b."}, "author_response": "We sincerely appreciate the reviewers' valuable comments and suggestions, which have greatly enhanced the quality of our manuscript. In response to your feedback, we have thoroughly revised the manuscript. Additionally, for the convenience of the reviewers, we have included a version of the manuscript highlighting the revised sections. The POINT-BY-POINT RESPONSES to the reviewers' com ments are as follows. The reviewer’s comm ents are in black and our responses a re in blue.\nReviewer #1\nRemarks to the Author:\nThe authors present an interesting investigation of the maternal factor for embryonic development known as NLRP14. Following up on a prior proteomic study, the authors first validate the ubiquitin ligases UHRF1 and KDM2a as prominent interactors of NLRP14. Using both cell-based assays and structural work, the authors find that NLRP14 is found to disrupt the localization of the E3s and alter UHRF1 protein levels, and present new structures of NLRP14 bound to either KDM2a or UHR1. These structures are biochemically validated using either binding or enzyme assays. Interestingly, NLRP14 blocks KDM2a’s ability to form a stable complex with the rest of the cullin-RING complex to form an active ubiquitin ligase. For UHRF1, NLRP14 binds to UHRF1’s domain that is needed for E2 recruitment, therefore, reducing its ability to ubiquitinate its targets. The data is of good quality and well presented. Overall, the authors should consider a few relatively minor points discussed below.\nRESPONSES: We sincerely thank you for your positive evaluation of our work and for the constructive comments. We have carefully considered all the suggestions and addressed each point in detail. We appreciate the improvements to the paper based on this valuable feedback.\n1. After the structures are presented, the authors do not circle back to examine if their structure-guided mutants would recapitulate the earlier presented phenotypes, e.g., KDM2a nuclear localization or decreased UHRF1 levels in the Nlrp14 null. These experiments will demonstrate that the effects observed are directly related to the interactions in the solved structures.\nRESPONSES: Thank you for raising this constructive suggestion. In the past three months, we have struggled with the microinjection of rescue treatment. We generated and injected Gfp (as a control) and N-Strep-mNlrp14 mRNA into WT and Nlrp14null GV oocytes. Definitely, we observed high-level GFP expression in WT and Nlrp14null GV oocytes (Response Figure 1a). However, the expression of mNLRP14 in GV oocytes was low, even undetectable (Response Figure 1b). Therefore, we further generated and injected codon-optimized WT mNlrp14 and mNlrp14-mutant (F201A) (this mutation affects the interaction with UHRF1) mRNA. We still observed weak expression of WT mNLRP14 and mNLRP14-F201A in oocytes (Response Figure 1c). The fluorescence intensity of UHRF1 of the three groups did not change in Nlrp14null GV oocytes (Response Figure 1d). We suspect that the knockout of Nlpr14 has a multifaceted impact on oocyte quality and leads to irreversible changes in oocyte proteostasis by abnormal activation of the ubiquitin-proteasome system. Thus, sole supplementation of its mRNA in GV oocytes cannot rescue the functional defects, and also cannot determine the impact of the NLRP14 mutant in vivo. Therefore, we just present this preliminary data in our response.\n2. In the figure 4, how are there more proteasomes or increase the size of the proteasomes? This result seems like a surprising finding unless proteasome assemblies are changing and their expression patterns are altered. Or are the authors stating that these are more aggregated proteasomes? Please expand and clarify what is happening at the molecular level. Furthermore, it is unclear if these results are directly due to the E3s discussed in the paper or the other ubiquitination machinery brought up in the discussion.\nRESPONSES: Thank you for raising this important point. We have revised the manuscript (line 328-333) and updated Figure 4 to clarify the nature of the “aggregated proteasomes”. The increase in both the number and size of proteasome puncta does not reflect an assembly of larger proteasome complexes. Instead, our data are consistent with the formation of ELVAs (endolysosomal vesicular assemblies, Zaffagnini et al. Cell 2024 PMID: 38382525), which co-concentrate ubiquitinated protein aggregates, lysosomes, and proteasomes. In Nlrp14-deficient oocytes, we observe concomitant increases in ubiquitinated proteins, lysosomal markers, and proteasomal signals, suggesting that the enhanced proteasome signal reflects the expansion of ELVAs rather than proteasome assemblies.\n3. E2s are not considered ubiquitin ligases. Also calling them “UBE2s” is awkward and confusing as the last letter is used to specify the E2. Please replace with Ubiquitin-conjugating enzymes or E2s or E2 enzymes. Additionally, in the results, the authors shouldn’t overstate that the Ubl mechanism is broadly applicable to all E2s that may be used by UHRF1. It is unclear if E2s other than UBE2D use the Ubl for their mechanism, assuming UHRF1 uses other E2s.\nRESPONSES: Thank you for pointing out this mistake. All “E2s” were replaced by specific UBE2D in the revised manuscript. According to two reports, UBE2D is the coordinated E2 ubiquitin-conjugating enzyme for the UBL domain of UHRF1 (DaRosa, P.A. et al. Mol Cell 2018 PMID: 30392931; Foster, B.M. et al. Mol Cell 2018 PMID: 30392929). To avoid overstatement, we have revised the relevant statements to clearly limit our conclusion to the E2 ubiquitin-conjugating enzyme tested in this study (UBE2D) and now describe the broader applicability to other E2 ubiquitin-conjugating enzymes as a possibility that remains to be determined.\n4. The last paragraph of the abstract can be expanded to summarize their paper.\nRESPONSES: Point taken. We have expanded it in the revised Abstract.\n5. The authors make this statement “A recent study on Nlrp14null mice revealed that the essential transporter NCXL on mitochondria dramatically decreased via the ubiquitylation-proteasome degradation pathway21, supporting the notion that NLRP14 inhibited the E3 activity in oocytes and embryos.“ However, unless there is evidence that is kdm2a or uhrf1 dependent, this statement seems out of place and may not be relevant.\nRESPONSES: Thank you for pointing out this mistake. We have deleted this sentence in the revised manuscript.\nReviewer #2\nRemarks to the Author:\nThe presented article is based on well-conducted, state-of-the-art experiments, provides novel insights into protein degradation during oocyte maturation, which represents an actual topic. The experimental data are appropriately complemented by structural prediction of interacting proteins and many extended data. I recommend this article for publication. However, prior to publication, the following minor issues should be addressed appropriately.\nRESPONSES: We sincerely thank you for your positive assessment of our work and for the recommendation for publication. We are pleased you find the study novel and well-conducted. We have carefully addressed all the minor points raised and incorporated the suggested improvements in the revised manuscript.\n1. The Discussion section should place greater emphasis on the obtained results. For example, the structure of proteins and complexes is not addressed at all, even though it constitutes a substantial portion of the Results section.\nRESPONSES: Thank you for your constructive suggestion. We have revised the discussion and added the points you mentioned (line 346-412).\n2. Line 398 – 400 – It is not appropriate to describe the mentioned proteins as critical structural components of CPLs. These proteins are rather stored on CPLs. Please revise this statement accordingly.\nRESPONSES: Point taken. We have corrected the sentence in the revised manuscript.\n3. Line 378 – Please specify which tubulin proteins you are referring to.\nRESPONSES: Thank you for your constructive suggestion. We have corrected this sentence with additional details in this revision.\n4. Lines 123 – 125. The statement “human NLRP14 only maintains the capacity to pull down the N-terminal of human KDM2B” is confusing. Specify what is the difference between the interaction of human NLRP14 and KDM2A/B and murine NLRP14 and KDM2A/B.\nRESPONSES: Thank you for raising this important point. Definitely, Pull-down assays show that human NLRP14 just interacts with human KDM2B (the C-terminal, 966-1336 aa), instead of “the N-terminal of human KDM2B”. We have corrected this sentence in this revision.\n5. Line 315 – Please define “their” as it is not clear what it refers to.\nRESPONSES: Thank you for pointing this out. We have rephrased this sentence.\n6. Line 335 – Please specify which process you are referring to.\nRESPONSES: Thank you for pointing this out. The process refers to “the increased protein ubiquitination”.We realize that this sentence is easily misunderstood and have deleted it.\n7. The overall quality of English is good. Nevertheless, in several places it requires improvement as the inaccurate formulations reduce the quality of the paper and sometimes hinder comprehension (e.g. incorrect use of on/in changing the meaning, omission of was in the passive voice, or confusion between removement/removal and others).\nRESPONSES: We sincerely thank you for this critical and helpful comment. We apologize for these oversights and have undertaken a comprehensive language revision.\n8. The questions included in the Results section (lines 170 – 171 and 213 – 215) are disruptive and do not integrate well with the surrounding text. Please consider rephrasing of these sentences.\nRESPONSES: Point taken. We have revised these sentences.\n9. Please explain all abbreviation used in the manuscript, including those that are commonly known (e.g. PDB, RMSD, IF and others).\nRESPONSES: Thank you for your constructive suggestion. We have explained all abbreviations in the revised manuscript.\n10. In the Methods section, several formulations remain written in a protocol-like style. Please revise these sentences to ensure they are appropriate in the Methods section. Line 583: are/were, Line 677/678 – whole sentence: Collect 10 ovaries from…..\nRESPONSES: Thank you for your suggestions. We have rephrased these sentences in this revision.\n11. Line 697: The citation Melina et al., 2023 is not formatted as a numerical reference and is missing from the References list.\nRESPONSES: Thank you for your constructive suggestion. We apologize for the inadequate citation. We have corrected it in this revision.\n12. Line 192 – typing error – analyse/analysed\nRESPONSES: Thank you for pointing this out. We have corrected this word in the revised manuscript.\nLine 346 – typing error – nuclus/nucleus\nRESPONSES: Thank you for pointing this out. We have corrected it.\nReviewer #3 (Remarks to the Author):\nThe manuscript from the Deng lab at Sichuan University (Chengdu) describes the molecular mechanisms by which the maternal factor NLRP14 regulates early embryonic development in mammals. The authors find that NLRP14 modulates protein homeostasis during the oocyte-to-embryo transition by interacting with two types of E3 ubiquitin ligases: the SCF (SKP1–CUL1–F-box) complex and the RING-type ligase UHRF1. Using biochemical assays and cryo-EM, they show that NLRP14 inhibits SCF complex formation by competing with CUL1 for KDM2A binding. NLRP14 also blocks UHRF1’s ubiquitination activity by preventing E2 ligase recruitment to its UBL domain. In Nlrp14-deficient mice, this regulatory loss led to excessive protein ubiquitination, disrupted proteostasis, and early embryonic arrest. The findings reveal that NLRP14 safeguards protein stability during oocyte maturation and fertilization, offering new insight into maternal control of proteostasis and reproductive disorders.This is a comprehensive study on the structure and function of NLRP14 in oocytes. The manuscript builds in part on a previous study from a different lab, where proteomic analysis identified 265 proteins that interact with NLRP14 in mouse oocytes (cited as reference 21). The manuscript is somewhat tedious to read, partly due to its verbose language and cumbersome explanations. This holds particularly for the structural descriptions. However, the topic of the study is interesting and timely, adding to our understanding of the oocyte-to-embryo transition, and the protein complexes analyzed are of importance.\nRESPONSES: We appreciate your encouraging comments on the comprehensiveness and relevance of our findings. We sincerely apologize for the tedious reading experience caused by verbose language. In our revision, we focused on streamlining the manuscript throughout and completely rewrote the structural description sections to make them more concise and accessible. Our goal is to ensure that the manuscript clearly conveys the novel mechanism insights.\nCriticism\n1. Albeit lengthy, the description and presentation of the cryo-EM structures is not very precise and not according to the standards in structural biology. When the authors introduce recombinant formation of the NLRP14–KDM2A–SKP1 complex, the authors speak of a “noncanonical peak with a shoulder” (line 182). What is this and at which expected size is the peak eluting? The authors continue describing that they determined the structures of “NLRP14-KDM2A-SKP1 in two distinct oligomeric states” (line 186) but then describe the first one only while the second one is introduced later in line 201. It would more to the point to describe the main peak as a heterotrimer and the shoulder left of it as a dimer of this heterotrimer.\nRESPONSES: Thank you for pointing this out. Firstly, we acknowledge that the description of the cryo-EM structures lacks precision, and we have thoroughly revised this section to ensure it meets the established standards in structural biology. (line 171-223). Secondly, “noncanonical peak with a shoulder” refers to the SEC profile of the purified NLRP14–KDM2A–SKP1 complex, which shows a main peak (Peak1) and a shoulder to its left (Peak2). In fact, the Peak1 eluting at 16 mL forms a heterotrimer complex and the Peak2 eluting at 14.5 mL assembles into a dimer of heterotrimers. Finally, as your suggestion, we have corrected our description of “monomer” and “dimer” into “heterotrimer” and “dimer of heterotrimers”.\n2. How is the dimer of the ternary NLRP14–KDM2A–SKP1 complex formed? This is not well described in the paragraph starting at line 201 and also not well visible in Fig. 2g and h. I assume, it is a C2 symmetry? The authors say that the dimer formed of two trimers is “… indicating the dynamic equilibration feature of NLRP14-KDM2A-SKP1 in solution …” (line 203). Where is this shown in the Ext. Data Fig. 7a? Is this dynamic equilibrium a result of a post-translational modification or is it concentration dependent? The authors should show size exclusion chromatograms similar to Fig. 2d at varying complex concentrations in order to specify if the observed splitting in a shoulder and main peak is a concentration dependent dynamic equilibrium. If the splitting would be dependent on a PTM, that occurs for example during the expression, the authors should perform comparative peptide mass finger-print analysis, e.g. from the fractions shown in ED Fig. 5b.\nRESPONSES: We appreciate your insightful question regarding the dimerization of the NLRP14-KDM2A-SKP1 ternary complex. First of all, we support the dimer of NLRP14-KDM2A-SKP1 adopts a pseudo-C2 symmetric arrangement. As illustrated in the attached local resolution map (Response Figure 2a), which was obtained from refinement in C1 symmetry, one heterotrimer exhibits higher local resolution, while the other displays relatively lower resolution. This asymmetry in map quality suggests that the dimer does not conform to a strict, ideal C2 symmetry and instead reflects inherent flexibility at the dimeric interface. Initially, we attempted to obtain a high-resolution density from C2-symmetry refinement. However, it showed a poorer resolution of 3.5 Å (Response Figure 2b-c). Therefore, we performed additional refinement using the local mask strategy, which yields clearer density for further analysis. Furthermore, we interpret this structural flexibility as a plausible molecular explanation for the dynamic equilibrium behavior observed for the NLRP14-KDM2A-SKP1 complex in solution. As we assumed the observed splitting in a shoulder and main peak is a concentration-dependent dynamic equilibrium, we re-injected the dimer of heterotrimers fraction (elution volume: 14.5 mL). As a result, the peak was partially shifted to the position corresponding to the heterotrimers (16 mL), indicating the dynamic equilibration feature of NLRP14-KDM2A-SKP1 in solution (Response Figure 2d). Additionally, the concentration-gradient protein cross-linking experiment revealed that oligomerization of the complex increased with increasing protein concentration, suggesting that NLRP14-KDM2A-SKP1 dimerization is concentration-dependent (Response Figure 2e). On the other hand, we did not observe any additional modification density at the dimer interface, suggesting that it is unlikely to undergo PTM modification regulation.\n3. In Extended Data Figure 7 c and d, the subunits are not labelled. As an intermolecular contact, shouldn’t, e.g., R357 be labelled as R357’ according to the standards?\nRESPONSES: Thank you for pointing this out. We have added these annotations to make the image easier to understand.\n4. The dimer formation of the NLRP14-KDM2A-SKP1 complex should be shown from a better perspective. Either in ED Fig. 7a, b or in Fig. 2.\nRESPONSES: Thank you for raising this suggestion. Three views of the dimer are shown in Extended Figure7 in this revision.\n5. Line 205f: “… the dimeric interface of NLRP14-KDM2A-SKP1 suggests two pairs of polar contacts (E462 and K959) …” In which subunit(s)? Please specify.\nRESPONSES: Thank you for pointing this out. We have improved this sentence.\n6. Line 207f: “Interestingly, the NLRP14 variants E462 and K959 cannot disrupt the dimer, …” Which variants? I can identify the mutants E462A and K959A marked in ED Fig. 7e.\nRESPONSES: Thank you for pointing this out. We apologize for the incorrect statement and we have improved this sentence.\n7. While I could continue here, the entire description of the complex structures should be substantially revised and improved.\nRESPONSES: Thank you for this critical feedback. We acknowledge that the description requires major improvement. In this revision, we focus on the following key actions to substantially enhance this section (line 171-223, 258-277): Enriching Structural Details: We had added precise descriptions of key structural features to provide a more complete and insightful analysis. Streamlining the Narrative: We had condensed and streamlined the language to improve readability and focus. Ensuring Precision and Professionalism: We rigorously revised the manuscript, employed accurate terminology and elevated the overall language to meet professional standards.\n8. The mutually exclusive binding properties between NLRP14 and UBL compared to UBE2D2 and UBL could be described a bit better and in other terms (‘mutually exclusive’, ‘displacement’). Can the authors determine the dissociation constants of these interactions?\nRESPONSES: Thank you for pointing this out. We avoid describing it as \"competitive\" in the revised manuscript. In addition, we also measured the binding affinities of NLRP14-UBL and UBE2D2-UBL via ITC (shown below). As shown in the result, the Kd for NLRP14 and UBL is 1.77 uM. However, the binding affinity between UBE2D2 and UBL is undetectable under identical conditions, although UBL can pull down UBE2D2 in an overexpression system (Fig. 3h).\n9. It is surprising that the cryo-EM structure determination of NLRP14 in two different complexes is not mentioned in the abstract, despite two figures in the main text describing the structural analysis, as also most of the extended data figures do and the ED table. Please correct this.\nRESPONSES: We sincerely thank you for this insightful comment. We have revised the abstract to appropriately emphasize the cryo-EM findings.\n10. Abstract, line 47: “… thereby establishing a theoretical framework for further investigation …” What does ‘theoretical framework’ mean. Please specify.\nRESPONSES: Thank you for pointing this out. This study shows that NLRP14, a factor involved in early mouse embryogenesis, plays a critical role in maintaining the protein degradation system in oocytes. Dysfunction of NLRP14 disrupts this system, adversely affecting oocyte development and embryo formation. The identified interaction partners and this mechanistic insight offer a valuable theoretical basis for clinical diagnosis. In the revised manuscript, we rewrote the abstract.\n11. The figure legends are rather lengthy, maybe they can be shortened.\nRESPONSES: We thank you for raising this suggestion. We have streamlined them in the revised manuscript.\n12. Discussion: The paragraph starting in line 389 (“Proteostasis is critical …”) is very good and describes the rational of the study. I wonder, if this could be a good starting paragraph for the discussion section, to provide some more background to the reader. By the way, “Proteins in oocytes exhibit an exceptionally long lifespan, with half-lives significantly exceeding those found in other cell types51.” (line 391). Can the authors specify the lifespan in terms of time?\nRESPONSES: Thank you for raising a valuable point. First of all, we have expanded to clarify this in the revised manuscript. In the previous report (Harasimov, K. et al. Nat Cell Biol 2024 PMID: 38902423), the \"exceptionally long lifespan\" refers to proteins with half-lives often exceeding 100 days, with a specific subset averaging roughly 681 days, effectively lasting for the organism's lifetime. In the ovary, over 10% of the proteins had half-lives exceeding 100 days, which is significantly higher than in other tissues, such as the brain, muscle, and cartilage.\n13. The model Figure 5 is not very compelling. Could the authors put this into a bigger picture, particularly with respect to the importance of protostasis in oocytes as described in the discussion section (see above). The essence of this study, that NLRP14 inhibits ubiquitination activity by interacting with two different E3 ligase complexes, should be emphasized more strongly in such a cartoon.\nRESPONSES: Thank you for this valuable comment. The revised Figure 5 is provided in the revised manuscript.\nMinor\n14. Line 56,57: “Mutations in NLRPs are associated with female reproductive diseases4,7,8.” You may change diseases to ‘disorders’ or ‘dysfunctions’.\nRESPONSES: Point taken. We have corrected these sentences in the revised manuscript.\n15. Line 123f: “Interestingly, human NLRP14 only maintains the capacity to pull down the N-terminal of human KDM2B, …” This sentence is not clear to me.\nRESPONSES: Point taken.\n16. Line 193: “monomeric NLRP14-KDM2A-SKP1 complex.” might be changed to ‘globular’, ‘heterotrimeric’ or ‘ternary’.\nRESPONSES: Point taken.\n17. Line 313: “Therefore, all these ‘observations’ suggest that …“\nRESPONSES: Point taken.\n18. The degree of rotation in Fig. 3c should be specified (I assume 90).\nRESPONSES: Point taken.\n19. Supplements, line 23: The conserved interaction …\nRESPONSES: Point taken.\n20. Supplements, line 41: The conserved interaction …\nRESPONSES: Point taken.\n21. Supplements, line 110: Residues mediating interactions between XXX and XXX are shown. I am unable to align the displays in panels c and d with the boxes in panel b.\nRESPONSES: Thank you for this valuable comment. We have corrected it in this revision."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my concerns.", "reviewer_3": "The authors present a substantially revised version of their original article. The description of protein structures has been improved, but still lacks in part precision and clarity.\nPlease correct the statement in line 218: “However, the NLRP14 variants E462A and K959A did not disrupt the dimer, suggesting that electrostatic interactions played a significant role in dimer formation (Extended Data Fig. 7f).” If removal of the charge does not disrupt complex formation, electrostatic association cannot play a significant role. In the previous version this sentence was (line 207): “Interestingly, the NLRP14 variants E462 and K959 cannot disrupt the dimer, also supporting the notion that van der Waals interactions play a significant role in dimer formation (Extended Data Fig. 7e).” The FLAG pull down in ED Fig. 7f shows a significantly diminished band for NLRP14_HA in the E462A lane and a slightly diminished band for the K959A variant. Therefore both, electrostatic and van der Waals interactions seem to play a role in complex formation."}, "author_response": ""}]

Decision letter