Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain lysine-specific demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and dimethylated H3-K4 of nucleosomes. Here, we report the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosome binding modules.

Two Gram-stain negative, catalase positive, coccus shaped bacteria, designated 10023T and 10010, were isolated from the rectal contents of a plateau pika (Ochotona curzoniae) in Qinghai-Tibet Plateau, China. Based on 16S rRNA gene sequence analysis, phylogenetic trees showed that these two isolates (10023T, 10010) group with members of the genus Neisseria. Additionally, these two isolates exhibited high 16S rRNA gene sequence similarity with Neisseria zalophi CSL 7565T (96.98%), Neisseria wadsworthii WC 05-9715T (96.92%) and Neisseria canis ATCC 14687T (96.79%). Further phylogenetic analysis based on the rplF gene showed that these two novel strains can be easily discriminated from phylogenetically closely related species. Optimal growth was found to occur on BHI agar with 5% defibrinated sheep blood at 37 °C and growth was also observed on nutrient agar, Columbia blood agar and chocolate agar plates; however, growth was not observed on MacConkey agar after 7 days. The major cellular fatty acids of these strains were identified as C16:0 and C16:1ω7c/C16:1ω6c. The complete genome size of the type strain 10023T is 2,496,444 bp, with DNA G+C content of 54.0 mol %. The average nucleotide identity values were 73.5-79.3% between isolate 10023T and reference Neisseria spp. Based on polyphasic analysis, these isolates (10023T and 10010) are considered to represent a novel species in the genus Neisseria, for which the name Neisseria chenwenguii sp. nov. is proposed. The type strain is 10023T (= DSM 103440T = CGMCC 1.15736T).

The Mst-Lats kinase cascade is central to the Hippo tumor-suppressive pathway that controls organ size and tissue homeostasis. The adaptor protein Mob1 promotes Lats activation by Mst, but the mechanism remains unknown. Here, we show that human Mob1 binds to autophosphorylated docking motifs in active Mst2. This binding enables Mob1 phosphorylation by Mst2. Phosphorylated Mob1 undergoes conformational activation and binds to Lats1. We determine the crystal structures of phospho-Mst2-Mob1 and phospho-Mob1-Lats1 complexes, revealing the structural basis of both phosphorylation-dependent binding events. Further biochemical and functional analyses demonstrate that Mob1 mediates Lats1 activation through dynamic scaffolding and allosteric mechanisms. Thus, Mob1 acts as a phosphorylation-regulated coupler of kinase activation by virtue of its ability to engage multiple ligands. We propose that stepwise, phosphorylation-triggered docking interactions of nonkinase elements enhance the specificity and robustness of kinase signaling cascades.

Boosting and prolonging SARS-CoV-2 vaccine-elicited immunity is paramount for containing the COVID-19 pandemic, which wanes substantially within months after vaccination. Here we demonstrate that the unique strain of probiotic Lactobacillus plantarum GUANKE (LPG) could promote SARS-CoV-2-specific immune responses in both effective and memory phases through enhancing interferon signaling and suppressing apoptotic and inflammatory pathways. Interestingly, oral LPG administration promoted SARS-CoV-2 neutralization antibodies even 6 months after immunization. Furthermore, when LPG was given immediately after SARS-CoV-2 vaccine inoculation, specific neutralization antibodies could be boosted >8-fold in bronchoalveolar lavage (BAL) and >2-fold in sera, T-cell responses were persistent and stable for a prolonged period both in BAL and the spleen. Transcriptional analyses showed that oral application of LPG mobilized immune responses in the mucosal and systemic compartments; in particular, gut-spleen and gut-lung immune axes were observed. These results suggest that LPG could be applied in combination with SARS-CoV-2 vaccines to boost and prolong both the effective and memory immune responses in mucosal and systemic compartments, thereby improving the efficacy of SARS-CoV-2 vaccination.

During mitosis of human cells, the kinase Bub1 orchestrates chromosome segregation through phosphorylating histone H2A and the anaphase-promoting complex/cyclosome activator Cdc20. Bub1-mediated H2A-T120 phosphorylation (H2A-pT120) at kinetochores promotes centromeric sister-chromatid cohesion, whereas Cdc20 phosphorylation by Bub1 contributes to spindle checkpoint signaling. Here, we show that phosphorylation at the P+1 substrate-binding loop of human Bub1 enhances its activity toward H2A but has no effect on its activity toward Cdc20. We determine the crystal structure of phosphorylated Bub1. A comparison between structures of phosphorylated and unphosphorylated Bub1 reveals phosphorylation-triggered reorganization of the P+1 loop. This activating phosphorylation of Bub1 is constitutive during the cell cycle. Enrichment of H2A-pT120 at mitotic kinetochores requires kinetochore targeting of Bub1. The P+1 loop phosphorylation of Bub1 appears to occur through intramolecular autophosphorylation. Our study provides structural and functional insights into substrate-specific regulation of a key mitotic kinase and expands the repertoire of kinase activation mechanisms.

In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C(Cdc20) inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C) or asymmetric O-Mad2-C-Mad2 (O-C) dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

Antioxidant peptides have been widely reported, whereas the intracellular antioxidant activity of a tripeptide (Pro-His-Pro, PHP), which was newly isolated and identified from Chinese Baijiu in our previous study, are still poorly understood. This study investigated the protective effects of PHP on 2,2'-azobis (2-methyl-propanimidamidine) dihydrochloride (AAPH)-induced oxidative stress in HepG2 cells and the involved molecular mechanisms. Pretreatment with PHP suppressed the generations of reactive oxygen species (ROS), malondialdehyde (MDA) and oxidized glutathione (GSSG), prevented a decrease in reduced glutathione (GSH), and up-regulated the activities of cellular antioxidant enzymes. Moreover, PHP treatment stimulated the mRNA and protein expression levels of antioxidant enzymes and nuclear factor E2 related factor 2 (Nrf2). Meanwhile, PHP markedly reduced the level of Kelch-like ECH-associated protein 1 (Keap1), suggesting that PHP effectively activated Nrf2/antioxidant response element (ARE)-mediated activity. These findings provide the first molecular basis for the health-promoting effects of PHP to prevent AAPH-induced oxidative stress.

The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1-4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.

The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1-MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

Microtubules are regulated by post-translational modifications of tubulin. The ligation and cleavage of the carboxy-terminal tyrosine of α-tubulin impact microtubule functions during mitosis, cardiomyocyte contraction and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1-SVBP alone, in complex with a tyrosine-derived covalent inhibitor and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α-tubulin tail by VASH1. The VASH1-SVBP-parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

ADAMTS12, a member of the ADAMTS family, is reported to be associated with the clinic outcome of colorectal cancer (CRC) patients. However, the functions and precise mechanism in CRC progression have yet to be fully understood.

The MST-LATS kinase cascade is central to the Hippo pathway that controls tissue homeostasis, development, and organ size. The PP2A complex STRIPAKSLMAP blocks MST1/2 activation. The GCKIII family kinases associate with STRIPAK, but the functions of these phosphatase-associated kinases remain elusive. We previously showed that the scaffolding protein SAV1 promotes Hippo signaling by counteracting STRIPAK (Bae et al., 2017). Here, we show that the GCKIII kinase STK25 promotes STRIPAK-mediated inhibition of MST2 in human cells. Depletion of STK25 enhances MST2 activation without affecting the integrity of STRIPAKSLMAP. STK25 directly phosphorylates SAV1 and diminishes the ability of SAV1 to inhibit STRIPAK. Thus, STK25 as the kinase component of STRIPAK can inhibit the function of the STRIPAK inhibitor SAV1. This mutual antagonism between STRIPAK and SAV1 controls the initiation of Hippo signaling.

Rarely has the vast diversity of bacteria on Earth been profiled, particularly on inaccessible plateaus. These uncultured microbes, which are also known as "microbial dark matter," may play crucial roles in maintaining the ecosystem and are linked to human health, regarding pathogenicity and prebioticity. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is a keystone species in the maintenance of ecological balance. We used a combination of full-length 16S rRNA amplicon sequencing, shotgun metagenomics, and metabolomics to elucidate the species-level community structure and the metabolic potential of the gut microbiota of the plateau pika. Using a full-length 16S rRNA metataxonomic approach, we clustered 618 (166 ± 35 per sample) operational phylogenetic units (OPUs) from 105 plateau pika samples and assigned them to 215 known species, 226 potentially new species, and 177 higher hierarchical taxa. Notably, 39 abundant OPUs (over 60% total relative abundance) are found in over 90% of the samples, thereby representing a "core microbiota." They are all classified as novel microbial lineages, from the class to the species level. Using metagenomic reads, we independently assembled and binned 109 high-quality, species-level genome bins (SGBs). Then, a precise taxonomic assignment was performed to clarify the phylogenetic consistency of the SGBs and the 16S rRNA amplicons. Thus, the majority of the core microbes possess their genomes. SGBs belonging to the genus Treponema, the families Muribaculaceae, Lachnospiraceae, and Oscillospiraceae, and the order Eubacteriales are abundant in the metagenomic samples. In addition, multiple CAZymes are detected in these SGBs, indicating their efficient utilization of plant biomass. As the most widely connected metabolite with the core microbiota, tryptophan may relate to host environmental adaptation. Our investigation allows for a greater comprehension of the composition and functional capacity of the gut microbiota of the plateau pika. IMPORTANCE The great majority of microbial species remain uncultured, severely limiting their taxonomic characterization and biological understanding. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is considered to be the keystone species in the maintenance of ecological stability. We comprehensively investigated the gut microbiota of the plateau pika via a multiomics endeavor. Combining full-length 16S rRNA metataxonomics, shotgun metagenomics, and metabolomics, we elucidated the species-level taxonomic assignment of the core uncultured intestinal microbiota of the plateau pika and revealed their correlation to host nutritional metabolism and adaptation. Our findings provide insights into the microbial diversity and biological significance of alpine animals.

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

Intestinal homeostasis is tightly regulated by complex yet poorly understood signaling networks. Here, we demonstrate that Lats1/2, the core Hippo kinases, are essential to maintain Wnt pathway activity and intestinal stem cells. Lats1/2 deletion leads to loss of intestinal stem cells but drives Wnt-uncoupled crypt expansion. To explore the function of downstream transcriptional enhanced associate domain (TEAD) transcription factors, we identified a selective small-molecule reversible inhibitor of TEAD auto-palmitoylation that directly occupies its lipid-binding site and inhibits TEAD-mediated transcription in vivo. Combining this chemical tool with genetic and proteomics approaches, we show that intestinal Wnt inhibition by Lats deletion is Yes-associated protein (YAP)/transcriptional activator with PDZ-binding domain (TAZ) dependent but TEAD independent. Mechanistically, nuclear YAP/TAZ interact with Groucho/Transducin-Like Enhancer of Split (TLE) to block Wnt/T-cell factor (TCF)-mediated transcription, and dual inhibition of TEAD and Lats suppresses Wnt-uncoupled Myc upregulation and epithelial over-proliferation in Adenomatous polyposis coli (APC)-mutated intestine. Our studies highlight a pharmacological approach to inhibit TEAD palmitoylation and have important implications for targeting Wnt and Hippo signaling in human malignancies.

The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers.

Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin, and by phosphorylation of both the enzyme and substrates. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here we report crystal structures of the separase protease domain from the thermophilic fungus Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study, mutating two securin residues in a conserved motif that partly matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin.

The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.

The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2-Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31comet-Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13-p31comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region.