Negative regulation of SH2B3 by SMYD5 controls epithelial-mesenchymal transition in lung cancer.

The main cause of death in lung cancer patients is metastasis. Thus, efforts to suppress micrometastasis or distant metastasis in lung cancer, identify therapeutic targets and develop related drugs are ongoing. In this study, we identified SET and MYND domain-containing protein 5 (SMYD5) as a novel metastasis regulator in lung cancer and found that SMYD5 was overexpressed in lung cancer based on both RNA-sequencing analysis results derived from the TCGA portal and immunohistochemical analysis results; knockdown of SMYD5 inhibited cell migration and invasion by changing epithelial-mesenchymal transition markers and MMP9 expression in NCI-H1299 and H1703 cell lines. Additionally, SMYD5 knockdown increased Src homology 2-b3 expression by decreasing the level of H4K20 trimethylation. Furthermore, in an in vitro epithelial-mesenchymal transition system using TGF-ß treatment, SMYD5 knockdown resulted in reduced cell migration and invasion in the highly invasive NCI-H1299 and H1703 cell lines. Based on these findings, we propose that SMYD5 could serve as a potential therapeutic target for lung cancer treatment and that cotreatment with an SMYD5 inhibitor and chemotherapy may enhance the therapeutic effect of lung cancer treatment.

Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids.

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling.

Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.

A bivalent form of a RBD-specific synthetic antibody effectively neutralizes SARS-CoV-2 variants.

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Structure-based drug discovery of a corticotropin-releasing hormone receptor 1 antagonist using an X-ray free-electron laser.

Thus far, attempts to develop drugs that target corticotropin-releasing hormone receptor 1 (CRF1R), a drug target in stress-related therapy, have been unsuccessful. Studies have focused on using high-resolution G protein-coupled receptor (GPCR) structures to develop drugs. X-ray free-electron lasers (XFELs), which prevent radiation damage and provide access to high-resolution compositions, have helped accelerate GPCR structural studies. We elucidated the crystal structure of CRF1R complexed with a BMK-I-152 antagonist at 2.75 Å using fixed-target serial femtosecond crystallography. The results revealed that two unique hydrogen bonds are present in the hydrogen bond network, the stalk region forms an alpha helix and the hydrophobic network contains an antagonist binding site. We then developed two antagonists-BMK-C203 and BMK-C205-and determined the CRF1R/BMK-C203 and CRF1R/BMK-C205 complex structures at 2.6 and 2.2 Å, respectively. BMK-C205 exerted significant antidepressant effects in mice and, thus, may be utilized to effectively identify structure-based drugs against CRF1R.

Anticancer Effects of Gut Microbiota-Derived Short-Chain Fatty Acids in Cancers.

Short-chain fatty acids (SCFAs), such as butyrate, propionate, and acetate produced by the gut microbiota have been implicated in physiological responses (defense mechanisms, immune responses, and cell metabolism) in the human body. In several types of cancers, SCFAs, especially butyrate, suppress tumor growth and cancer cell metastasis via the regulation of the cell cycle, autophagy, cancer-related signaling pathways, and cancer cell metabolism. In addition, combination treatment with SCFAs and anticancer drugs exhibits synergistic effects, increasing anticancer treatment efficiency and attenuating anticancer drug resistance. Therefore, in this review, we point out the importance of SCFAs and the mechanisms underlying their effects in cancer treatment and suggest using SCFA-producing microbes and SCFAs to increase therapeutic efficacy in several types of cancers.

Epigenetic regulation of DHRS2 by SUV420H2 inhibits cell apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC), also known as kidney cancer, is a common malignant tumor of the urinary system. While surgical treatment is essential, novel therapeutic targets and corresponding drugs for RCC are still needed due to the high relapse rate and low five-year survival rate. In this study, we found that SUV420H2 is overexpressed in renal cancers and that high SUV420H2 expression is associated with a poor prognosis, as evidenced by RCC RNA-seq results derived from the TCGA. SUV420H2 knockdown using siRNA led to growth suppression and cell apoptosis in the A498 cell line. Furthermore, we identified DHRS2 as a direct target of SUV420H2 in the apoptosis process through a ChIP assay with a histone 4 lysine 20 (H4K20) trimethylation antibody. Rescue experiments showed that cotreatment with siSUV420H2 and siDHRS2 attenuated cell growth suppression induced by SUV420H2 knockdown only. Additionally, treatment with the SUV420H2 inhibitor A-196 induced cell apoptosis via upregulation of DHRS2. Taken together, our findings suggest that SUV420H2 may be a potential therapeutic target for the treatment of renal cancer.

Oligomeric states of ASC specks regulate inflammatory responses by inflammasome in the extracellular space.

Inflammasomes are multi-protein complexes and play a crucial role in host defense against pathogens. Downstream inflammatory responses through inflammasomes are known to be related to the oligomerization degree of ASC specks, but the detailed mechanism still remains unexplored. Here, we demonstrate that oligomerization degrees of ASC specks regulate the caspase-1 activation in the extracellular space. A protein binder specific for a pyrin domain (PYD) of ASC (ASCPYD) was developed, and structural analysis revealed that the protein binder effectively inhibits the interaction between PYDs, disassembling ASC specks into low oligomeric states. ASC specks with a low oligomerization degree were shown to enhance the activation of caspase-1 by recruiting and processing more premature caspase-1 through interactions between CARD of caspase-1 (caspase-1CARD) and CARD of ASC (ASCCARD). These findings can provide insight into controlling the inflammasome-mediated inflammatory process as well as the development of inflammasome-targeting drugs.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Novel bispecific human antibody platform specifically targeting a fully open spike conformation potently neutralizes multiple SARS-CoV-2 variants.

Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.

TRIM40 is a pathogenic driver of inflammatory bowel disease subverting intestinal barrier integrity.

The cortical actin cytoskeleton plays a critical role in maintaining intestinal epithelial integrity, and the loss of this architecture leads to chronic inflammation, as seen in inflammatory bowel disease (IBD). However, the exact mechanisms underlying aberrant actin remodeling in pathological states remain largely unknown. Here, we show that a subset of patients with IBD exhibits substantially higher levels of tripartite motif-containing protein 40 (TRIM40), a gene that is hardly detectable in healthy individuals. TRIM40 is an E3 ligase that directly targets Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), an essential kinase involved in promoting cell-cell junctions, markedly decreasing the phosphorylation of key signaling factors critical for cortical actin formation and stabilization. This causes failure of the epithelial barrier function, thereby promoting a long-lived inflammatory response. A mutant TRIM40 lacking the RING, B-box, or C-terminal domains has impaired ability to accelerate ROCK1 degradation-driven cortical actin disruption. Accordingly, Trim40-deficient male mice are highly resistant to dextran sulfate sodium (DSS)-induced colitis. Our findings highlight that aberrant upregulation of TRIM40, which is epigenetically silenced under healthy conditions, drives IBD by subverting cortical actin formation and exacerbating epithelial barrier dysfunction.

Endothelial PTP4A1 mitigates vascular inflammation via USF1/A20 axis-mediated NF-κB inactivation.

AIMS: The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. METHODS AND RESULTS: In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1ß-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. CONCLUSION: Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

Characterization of signature trends across the spectrum of non-alcoholic fatty liver disease using deep learning method.

AIMS: The timely diagnosis of different stages in NAFLD is crucial for disease treatment and reversal. We used hepatocellular ballooning to determine different NAFLD stages. MAIN METHODS: We analyzed differentially expressed genes (DEGs) in 78 patients with NAFLD and in healthy controls from previously published RNA-seq data. We identified two expression types in NAFLD progression, calculated the predictive power of candidate genes, and validated them in an independent cohort. We also performed cancer studies with these candidates retrieved from the Cancer Genome Atlas. KEY FINDINGS: We identified 103 DEGs in NAFLD patients compared to healthy controls: 75 genes gradually increased or decreased in the NAFLD stage, whereas 28 genes showed differences only in NASH. The former were enriched in negative regulation and binding-related genes; the latter were involved in positive regulation and cell proliferation. Feature selection showed the gradual up- or down-regulation of 21 genes in NASH compared to controls; 18 were highly expressed only in NASH. Using deep-learning method with subset of features from lasso regression, we obtained reliable determination performance in NAFL and NASH (accuracy: 0.857) and validated these genes using an independent cohort (accuracy: 0.805). From cancer studies, we identified significant differential expression of several candidate genes in LIHC; 5 genes were gradually up-regulated and 6 showing high expression only in NASH were influential to patient survival. SIGNIFICANCE: The identified biomolecular signatures may determine the spectrum of NAFLD and its relationship with HCC, improving clinical diagnosis and prognosis and enabling a therapeutic intervention for NAFLD.

Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans.

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.

Catalytic DxD motif caged in Asx-turn and Met-aromatic interaction attenuates the pathogenic glycosylation of SseK2/NleB2 effectors.

Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto host crucial proteins, which allows adhesion to mammalian cells and modulates host cellular processes for pathogenesis. Escherichia coli NleB1, Citrobacter rodentium NleB, and Salmonella enterica SseK1/3 type III effectors fatally glycosyltransfer N-acetyl glucosamine (GlcNAc) from UDP-GlcNAc to arginine residues of death domain-containing proteins that regulate host inflammation, intra-bacterial proteins, and themselves, whose post-translational modification disrupts host immune functions and prolongs bacterial viability inside host cells. However, unlike the similar NleB1/SseK1/SseK3, E. coli NleB2 and S. enterica SseK2 show deficient GlcNAcylation and neither intra-bacterial glycosylation nor auto-glycosylation. Here, as the major factor in SseK2/NleB2 deficiency, we focused on the catalytic Asp-x-Asp (DxD) motif conserved throughout all O-/N-glycosyltransferases to coordinate Mn2+. All DxD motifs in apo-glycosyltransferases form Type-I-turns for binding Mn2+, similar to the ligand-bound DxD motif, whereas TcnA/SseK2/NleB2 DxD motifs form Asx-turns, which are unable to bind Mn2+. Interestingly, methionine of the NleB2 DMD motif forms triple Met-aromatic interactions, as found in age-associated diseases and tumor necrosis factor (TNF) ligand-receptor complexes. The NleB1 A222M mutation induces triple Met-aromatic interactions to steeply attenuate glycosylation activity to 3% of that in the wild type. Thus, the characteristic conformation of the DxD motif is essential for binding Mn2+, donors, and glycosylate targets. This explains why SseK2/NleB2 effectors with the DxD motif caged in the Asp-/Asn-turn (Asx-turn) and triple Met-aromatic interactions have lower glycosyltransferase activity than that of other fatal NleB1/SseK1/SseK3 toxins.

Limosilactobacillus reuteri DS0384 promotes intestinal epithelial maturation via the postbiotic effect in human intestinal organoids and infant mice.

Little is known about the modulatory capacity of the microbiota in early intestinal development. We examined various intestinal models that respond to gut microbial metabolites based on human pluripotent stem cell-derived human intestinal organoids (hIOs): physiologically relevant in vitro fetal-like intestine, intestinal stem cell, and intestinal disease models. We found that a newly isolated Limosilactobacillus reuteri strain DS0384 accelerated maturation of the fetal intestine using 3D hIO with immature fetal characteristics. Comparative metabolomic profiling analysis revealed that the secreted metabolite N-carbamyl glutamic acid (NCG) is involved in the beneficial effect of DS0384 cell-free supernatants on the intestinal maturation of hIOs. Experiments in an intestinal stem cell spheroid model and hIO-based intestinal inflamed model revealed that the cell-free supernatant from DS0384 comprising NCG promoted intestinal stem cell proliferation and was important for intestinal protection against cytokine-induced intestinal epithelial injury. The probiotic properties of DS0384 were also evaluated, including acid and bile tolerance and ability to adhere to human intestinal cells. Seven-day oral administration of DS0384 and cell-free supernatant promoted the intestinal development of newborn mice. Moreover, NCG exerted a protective effect on experimental colitis in mice. These results suggest that DS0384 is a useful agent for probiotic applications and therapeutic treatment for disorders of early gut development and for preventing intestinal barrier dysfunction.