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Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ 4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium , a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ 4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible. Steroid 5α-reductase 2 (SRD5A2), a testosterone metabolism enzyme, is implicated in human disease. Structural and biochemical analyses of PbSRD5A, a bacterial homolog, reveal SRD5A2 substrate binding pocket and provide framework for the design of new drugs targeting this enzyme.

The subcortical maternal complex (SCMC) plays a crucial role in early embryonic development. Malfunction of SCMC leads to reproductive diseases in women. However, the molecular function and assembly basis for SCMC remain elusive. Here we reconstituted mouse SCMC and solved the structure at atomic resolution using single-particle cryo-electron microscopy. The core complex of SCMC was formed by MATER, TLE6 and FLOPED, and MATER embraced TLE6 and FLOPED via its NACHT and LRR domains. Two core complexes further dimerize through interactions between two LRR domains of MATERs in vitro. FILIA integrates into SCMC by interacting with the carboxyl-terminal region of FLOPED. Zygotes from mice with Floped C-terminus truncation showed delayed development and resembled the phenotype of zygotes from Filia knockout mice. More importantly, the assembly of mouse SCMC was affected by corresponding clinical variants associated with female reproductive diseases and corresponded with a prediction based on the mouse SCMC structure. Our study paves the way for further investigations on SCMC functions during mammalian preimplantation embryonic development and reveals underlying causes of female reproductive diseases related to SCMC mutations, providing a new strategy for the diagnosis of female reproductive disorders. Here, using cryo-electron microscopy and functional assays in mouse oocytes and embryos, the authors delineate the composition of the subcortical maternal complex, showing that clinical variants associated with female reproductive diseases disrupt complex formation.

The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis. The subcortical maternal complex (SCMC) maintains cleavage of early embryos. Here the authors identify 14-3-3 as a component of the SCMC and show that phosphorylation of TLE6 contributes to 14-3-3 recruitment and stabilization, ensuring mitotic entry in mouse zygotes.

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.

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. The maternal effector NLRP14 regulates two E3 ubiquitin ligases via distinct mechanisms to prevent excessive ubiquitination and maintain protein homeostasis during the oocyte-to-embryo transition, thereby supporting successful early embryonic development.

The functionally conserved subcortical maternal complex (SCMC) is essential for early embryonic development in mammals. Reproductive disorders caused by pathogenic variants in NLRP5, TLE6 and OOEP, three core components of the SCMC, have attracted much attention over the past several years. Evaluating the pathogenicity of a missense variant in the SCMC is limited by the lack of information on its structure, although we recently solved the structure of the mouse SCMC and proposed that reproductive disorders caused by pathogenic variants are related to the destabilization of the SCMC core complex. Here we report the cryogenic electron microscopy structure of the human SCMC and uncover that the pyrin domain of NLRP5 is essential for the stability of SCMC. By combining prediction of SCMC stability and in vitro reconstitution, we provide a method for identifying deleterious variants, and we successfully identify a new pathogenic variant of TLE6 (p.A396T). Thus, on the basis of the structure of the human SCMC, we offer a strategy for the diagnosis of reproductive disorders and the discovery of new infertility-associated variants. On the basis of the assembly mechanism and structures of the human subcortical maternal complex, the authors provide a strategy for the diagnosis of reproductive disorders and the discovery of new infertility-associated SCMC variants.

Bcl-2 (B-cell lymphoma 2), the first identified anti-apoptosis factor, encodes two transcripts, the long isoform α and the short isoform β . The current understanding of the Bcl-2 function mainly focuses on Bcl-2α, while little is known about the function of Bcl-2β, which lacks the transmembrane domain and contains 10 unique amino acids at the C-terminus instead. Here, we analyzed the expressions of BCL-2 two isoforms in diffused large B-cell lymphoma (DLBCL) and found a significant positive correlation between them. Then, with the CRISPR/Cas9-based transcriptional activator (CRISPRa), we generated mouse B-cell lymphomas with Bcl-2 upregulation from the endogenous locus, in which both Bcl-2α and Bcl-2β levels were increased. Bcl-2β itself promoted angiogenesis both in vitro and in vivo through increased vascular endothelial growth factor A (VEGF-A). Inhibiting VEGF receptors with Axitinib reduced angiogenesis induced by Bcl-2β overexpression. Co-immunoprecipitation and mass spectrometry analysis revealed that Bcl-2β interacted with the T-complex protein ring complex (TRiC). Disruption of TRiC significantly impaired the angiogenesis-promoting activity of Bcl-2β, indicated by reduced VEGF-A protein level and HUVEC tube formation. Thus, our study suggests that Bcl-2 isoform β plays a role in promoting tumor angiogenesis through the Bcl-2β-TRiC-VEGF-A axis.

Small cell lung cancer (SCLC) is notorious for its early and frequent metastases, which contribute to it as a recalcitrant malignancy. To understand the molecular mechanisms underlying SCLC metastasis, we generated SCLC mouse models with orthotopically transplanted genome-edited lung organoids and performed multiomics analyses. We found that a deficiency of KMT2C, a histone H3 lysine 4 methyltransferase frequently mutated in extensive-stage SCLC, promoted multiple-organ metastases in mice. Metastatic and KMT2C-deficient SCLC displayed both histone and DNA hypomethylation. Mechanistically, KMT2C directly regulated the expression of DNMT3A, a de novo DNA methyltransferase, through histone methylation. Forced DNMT3A expression restrained metastasis of KMT2C-deficient SCLC through repressing metastasis-promoting MEIS/HOX genes. Further, S-(5'-adenosyl)-L-methionine, the common cofactor of histone and DNA methyltransferases, inhibited SCLC metastasis. Thus, our study revealed a concerted epigenetic reprogramming of KMT2C- and DNMT3A-mediated histone and DNA hypomethylation underlying SCLC metastasis, which suggested a potential epigenetic therapeutic vulnerability.

Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-delta-4 structure, such as testosterone, androstenedione, and progesterone, could be catalyzed by steroid 5-alpha reductases (SRD5As) to generate their corresponding 3-oxo-5-alpha steroids, which are essential for multiple physiological and pathological processes. Abnormal activities of SRD5As will lead to benign prostatic hyperplasia, alopecia, prostatic cancer, or infertility due to the poor quality of sperms. However, the detailed reduction mechanisms of SRD5As remain elusive. Here we report the crystal structure of PbSRD5A, which shares 60.6% and 51.5% sequence similarities with human SRD5A1 and -2 respectively, from Proteobacteria bacterium in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic cavity for steroids substrates binding, whereas TM5-7 coordinate with cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with extensive biochemical characterizations, for the first time unveiled the substrate recognition of SRD5As and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-delta-4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible. Competing Interest Statement The authors have declared no competing interest.