Jinsong Liu

The flexible structure enables nonstructural protein 8 (nsp8) to respond quickly to environmental changes, which are essential for RNA replication and transcription of SARS-CoV-2. In this work, small-angle neutron scattering and AlphaFold2 prediction were applied to characterize the structural change of SARS-CoV-2 nsp8 dimers and tetramers. The results demonstrated that the nsp8 tetramer with a more exposed core domain shows a low thermal stability. The exposed core domain increases its sensitivity to RNA and adapts its structure to interact with RNA. Our work reveals the structural difference between the two forms of SARS-CoV-2 nsp8s in the RNA synthesis process, which partly elucidates the molecular mechanism behind RNA replication of the RNA virus.

Polycomb repressive complexes (PRCs) are pivotal epigenetic regulators that preserve cell identity by restricting transcription responses to sub-threshold extracellular signals. Their roles in osteoblast function and bone formation remain unclear. Here in aging osteoblasts, we found marked activation of PRC1.1 complex, with KDM2B acting as a chromatin-binding factor and BCOR and PCGF1 enabling histone H2A monoubiquitylation (H2AK119ub1). Osteoblast-specific Kdm2b inactivation significantly enhances bone remodeling under steady-state conditions and in scenarios of bone loss. This enhancement is attributed to H2AK119ub1 downregulation and subsequent Wnt signaling derepression. Furthermore, we developed a small molecule termed iBP, that specifically inhibits the interaction between BCOR and PCGF1, thereby suppressing PRC1.1 activity. Notably, iBP administration promotes bone formation in mouse models of bone loss. Therefore, our findings identify PRC1.1 as a critical epigenetic brake on bone formation and demonstrate that therapeutic targeting of this complex enhances Wnt pathway activation, offering a promising strategy against skeletal deterioration.

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor in the ERR subfamily that plays a crucial role in regulating energy metabolism. To date, no endogenous ligand has been identified for ERRγ, posing a challenge for developing targeted therapeutics. Here, we identified that indole and skatole produced by the gut microbiota are potential endogenous ligands of ERRγ using biochemical, cellular, structural, and computational approaches. Indole and skatole increased ERRγ thermostability and directly bound to the ligand-binding domain (LBD) with a K_d of approximately 1–2 μM but had no significant effect or weak inhibitory activity on the transcriptional efficiency. However, RNA sequencing revealed that ERRγ could coregulate several lipid metabolism- and immune-related genes with indole, suggesting a role for ERRγ in the indole pathway. Interestingly, indole and skatole differentially attenuated the activities of ERRγ ligands: they both neutralized the agonistic activity of GSK4716, while indole reduced the antagonistic activity of 4-hydroxytamoxifen (4OHT) and GSK5182, and skatole affected the agonistic activity of endocrine disruptor bisphenol A (BPA). We further screened additional indole metabolites and analogs, resolved the complex structures of ERRγ-LBD with these compounds, and conducted molecular dynamics simulations to determine their binding site and elucidate their binding mechanisms. This study identified potential endogenous ligands of ERRγ, suggesting a novel link between the energy metabolism regulation and the indole pathway. Our findings highlight the need to consider endogenous ligands when designing and optimizing ERRγ-targeted drugs.

Farnesoid X receptor (FXR) is a promising target for treatment of metabolic associated fatty liver disease (MAFLD). In this study, we employed an integrative approach to investigate the interaction between FXR-RXRα-DNA complex and the entire coactivator SRC1-NRID (nuclear receptor interaction domain). We constructed a multi-domain model of FXR-RXRα-DNA, highlighting the interface between FXR-DBD and LBD. Using HDX-MS, XL-MS, and biochemical assays, we revealed the allosteric communications in FXR-RXRα-DNA upon agonist and DNA binding. We then demonstrated that SRC1 binds only to the coactivator binding surface of FXR within the FXR-RXRα heterodimer, with the NR-box2 and NR-box3 of SRC1 as the key binding motifs. Our findings, which provide the first model of SRC1-NRID in complex with FXR-RXRα-DNA, shed light on the molecular mechanism through which the coactivator asymmetrically interacts with nuclear receptors and provide structural basis for further understanding the function of FXR and its implications in diseases.

UHRF1 maintains DNA methylation by recruiting DNA methyltransferases to chromatin. In mouse, these dynamics are potently antagonized by a natural UHRF1 inhibitory protein STELLA, while the comparable effects of its human ortholog are insufficiently characterized, especially in cancer cells. Herein, we demonstrate that human STELLA (hSTELLA) is inadequate, while mouse STELLA (mSTELLA) is fully proficient in inhibiting the abnormal DNA methylation and oncogenic functions of UHRF1 in human cancer cells. Structural studies reveal a region of low sequence homology between these STELLA orthologs that allows mSTELLA but not hSTELLA to bind tightly and cooperatively to the essential histone-binding, linked tandem Tudor domain and plant homeodomain (TTD-PHD) of UHRF1, thus mediating ortholog-specific UHRF1 inhibition. For translating these findings to cancer therapy, we use a lipid nanoparticle (LNP)-mediated mRNA delivery approach in which the short mSTELLA, but not hSTELLA regions are required to reverse cancer-specific DNA hypermethylation and impair colorectal cancer tumorigenicity.

Polycomb repressive complexes (PRCs) are pivotal epigenetic regulators preserving cell identity by restricting the transcription responsiveness to sub-threshold levels of extracellular signals. Their roles in osteoblast function and bone formation remain largely unexplored. Here in aging osteoblasts, we observed a selective activation of PRC1.1 complex, with KDM2B acting as a chromatin-binding factor and BCOR and PCGF1 serving as essential catalytic partners for histone H2A monoubiquitylation (H2AK119ub1). Using genetic models, we found that osteoblast-specific KDM2B inactivation significantly enhances bone remodeling under steady-state conditions and in scenarios of bone loss. This enhancement is attributed to H2AK119ub1 downregulation which leads to the derepression of Wnt signaling. Furthermore, we developed a small molecule termed iBP, that specifically inhibits the interaction between BCOR and PCGF1, thereby suppressing PRC1.1 activity. Notably, iBP promotes bone formation in mouse models of bone loss. Therefore, our findings suggest that targeting PRC1.1 augments cellular responses to Wnt signaling and may offer a promising strategy to counteract bone deterioration.

Recruitment of non-canonical BCOR-PRC1.1 to non-methylated CpG islands via KDM2B plays a fundamental role in transcription control during developmental processes and cancer progression. However, the mechanism is still largely unknown on how this recruitment is regulated. Here, we unveiled the importance of the Poly-D/E regions within the linker of BCOR for its binding to KDM2B. Interestingly, we also demonstrated that these negatively charged Poly-D/E regions on BCOR play autoinhibitory roles in liquid-liquid phase separation (LLPS) of BCOR^{ANK-linker-PUFD}/PCGF1^RAWUL. Through neutralizing negative charges of these Poly-D/E regions, Ca²⁺ not only weakens the interaction between BCOR/PCGF1 and KDM2B, but also promotes co-condensation of the enzymatic core of BCOR-PRC1.1 with KDM2B into liquid-like droplet. Accordingly, we propose that Ca²⁺ could modulate the compartmentation and recruitment of the enzymatic core of BCOR-PRC1.1 on KDM2B target loci. Thus, our finding advances the mechanistic understanding on how the tethering of BCOR-PRC1.1 enzymatic core to KDM2B is regulated.

Farnesoid X receptor (FXR) is a promising target for treatment of metabolic associated fatty liver disease (MAFLD). In this study, we employed an integrative approach to investigate the interaction between FXR-RXRα-hSHP-1 complex and the entire coactivator SRC1-NRID (nuclear receptor interaction domain). We constructed a multi-domain model of FXR ₁₂₀ -RXRα ₉₈ -hSHP-1, highlighting the interface between FXR-DBD and LBD. Using HDX-MS, XL-MS, and biochemical assays, we revealed the allosteric communications in FXR-RXRα-hSHP-1 upon agonist and DNA binding. We then demonstrated that SRC1 binds only to the coactivator binding surface of FXR within the FXR-RXRα heterodimer, with the NR-box2 and NR-box3 of SRC1 as the key binding motifs. Our findings, which provide the first model of SRC1-NRID in complex with FXR-RXRα-hSHP-1, shed light on the molecular mechanism through which the coactivator asymmetrically interacts with nuclear receptors and provide structural basis for further understanding the function of FXR and its implications in diseases.

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (M ^pro ), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from M ^pro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 M ^pro :RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named ‘leritrelvir’ for COVID-19 treatment in China.