Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

Graphislactone A, a Fungal Antioxidant Metabolite, Reduces Lipogenesis and Protects against Diet-Induced Hepatic Steatosis in Mice.

Graphislactone A (GPA), a secondary metabolite derived from a mycobiont found in the lichens of the genus Graphis, exhibits antioxidant properties. However, the potential biological functions and therapeutic applications of GPA at the cellular and animal levels have not yet been investigated. In the present study, we explored the therapeutic potential of GPA in mitigating non-alcoholic fatty liver disease (NAFLD) and its underlying mechanisms through a series of experiments using various cell lines and animal models. GPA demonstrated antioxidant capacity on a par with that of vitamin C in cultured hepatocytes and reduced the inflammatory response induced by lipopolysaccharide in primary macrophages. However, in animal studies using an NAFLD mouse model, GPA had a milder impact on liver inflammation while markedly attenuating hepatic steatosis. This effect was confirmed in an animal model of early fatty liver disease without inflammation. Mechanistically, GPA inhibited lipogenesis rather than fat oxidation in cultured hepatocytes. Similarly, RNA sequencing data revealed intriguing associations between GPA and the adipogenic pathways during adipocyte differentiation. GPA effectively reduced lipid accumulation and suppressed lipogenic gene expression in AML12 hepatocytes and 3T3-L1 adipocytes. In summary, our study demonstrates the potential application of GPA to protect against hepatic steatosis in vivo and suggests a novel role for GPA as an underlying mechanism in lipogenesis, paving the way for future exploration of its therapeutic potential.

iCSDB: an integrated database of CRISPR screens.

High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.

Alterations in the Rho pathway contribute to Epstein-Barr virus-induced lymphomagenesis in immunosuppressed environments.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas (EBV+-DLBLs) tend to occur in immunocompromised patients, such as the elderly or those undergoing solid organ transplantation. The pathogenesis and genomic characteristics of EBV+-DLBLs are largely unknown because of the limited availability of human samples and lack of experimental animal models. We observed the development of 25 human EBV+-DLBLs during the engraftment of gastric adenocarcinomas into immunodeficient mice. An integrated genomic analysis of the human-derived EBV+-DLBLs revealed enrichment of mutations in Rho pathway genes, including RHPN2, and Rho pathway transcriptomic activation. Targeting the Rho pathway using a Rho-associated protein kinase (ROCK) inhibitor, fasudil, markedly decreased tumor growth in EBV+-DLBL patient-derived xenograft (PDX) models. Thus, alterations in the Rho pathway appear to contribute to EBV-induced lymphomagenesis in immunosuppressed environments.

Genomic alterations in BCL2L1 and DLC1 contribute to drug sensitivity in gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases.

Cystamine induces AIF-mediated apoptosis through glutathione depletion.

Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death.

Intimal hyperplasia in loop-injured carotid arteries is attenuated in transglutaminase 2-null mice.

Arterial restenosis frequently develops after open or endovascular surgery due to intimal hyperplasia. Since tissue transglutaminase (TG2) is known to involve in fibrosis, wound healing, and extracellular matrix remodeling, we examined the role of TG2 in the process of intimal hyperplasia using TG2-null mice. The neointimal formation was compared between TG2-null and wild-type (C57BL/6) mice by two different injury models; carotid ligation and carotid loop injury. In ligation model, there was no difference in intimal thickness between two groups. In loop injury model, intimal hyperplasia developed in both groups and the intimal/medial area ratio was significantly reduced in TG2-null mice (P = 0.007). TG2 was intensely stained in neointimal cells in 2 weeks. In situ activity of TG2 in the injured arteries steadily increased until 4 weeks compared to uninjured arteries. Taken together, intimal hyperplasia was significantly reduced in TG2-null mice, indicating that TG2 has an important role in the development of intimal hyperplasia. This suggests that TG2 may be a novel target to prevent the arterial restenosis after vascular surgery.

The Effect of Clostridium butyricum on Gut Microbial Changes and Functional Profiles of Metabolism in High-fat Diet-fed Rats Depending on Age and Sex.

Background/Aims: A high-fat diet (HFD) causes dysbiosis and promotes inflammatory responses in the colon. This study aims to evaluate the effects of Clostridium butyricum on HFD-induced gut microbial changes in rats. Methods: Six-week-old Fischer-344 rats with both sexes were given a control or HFD during 8 weeks, and 1-to-100-fold diluted Clostridium butyricum were administered by gavage. Fecal microbiota analyses were conducted using 16S ribosomal RNA metagenomic sequencing and predictive functional profiling of microbial communities in metabolism. Results: A significant increase in Ruminococcaceae and Lachnospiraceae, which are butyric acid-producing bacterial families, was observed in the probiotics groups depending on sex. In contrast, Akkermansia muciniphila, which increased through a HFD regardless of sex, and decreased in the probiotics groups. A. muciniphila positively correlated with Claudin-1 expression in males (P < 0.001) and negatively correlated with the expression of Claudin-2 (P = 0.042), IL-1ß (P = 0.037), and IL-6 (P = 0.044) in females. In terms of functional analyses, a HFD decreased the relative abundances of M00131 (carbohydrate metabolism module), M00579, and M00608 (energy metabolism), and increased those of M00307 (carbohydrate metabolism), regardless of sex. However, these changes recovered especially in male C. butyricum groups. Furthermore, M00131, M00579, and M00608 showed a positive correlation and M00307 showed a negative correlation with the relative abundance of A. muciniphila (P < 0.001). Conclusion: The beneficial effects of C. butyricum on HFD-induced gut dysbiosis in young male rats originate from the functional profiles of carbohydrate and energy metabolism.

Identification of New Pathogenic Variants of Hereditary Diffuse Gastric Cancer.

Purpose: Hereditary diffuse gastric cancer (HDGC) presents a significant genetic predisposition, notably linked to mutations in the CDH1 and CTNNA1. However, the genetic basis for over half of HDGC cases remains unidentified. The aim of this study is to identify novel pathogenic variants in HDGC and evaluate their protein expression. Materials and Methods: Among 20 qualifying families, two were selected based on available pedigree and DNA. Whole genome sequencing (WGS) on DNA extracted from blood and whole exome sequencing (WES) on DNA from formalin-fixed paraffin-embedded tissues were performed to find potential pathogenic variants in HDGC. After selection of a candidate variant, functional validation and enrichment analysis were performed. Results: As a result of WGS, three candidate germline mutations (EPHA5, MCOA2, and RHOA) were identified in one family. After literature review and in silico analyses, the RHOA mutation (R129W) was selected as a candidate. This mutation was found in two gastric cancer patients within the family. In functional validation, it showed RhoA overexpression and a higher GTP-bound state in the RhoaR129W mutant. Decreased phosphorylation at Ser127/397 suggested altered YAP1 regulation in the Rho-ROCK pathway. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses linked RhoAR129W overexpression to changed migration/adhesion in MKN1 cell line. However, this RHOA mutation (R129W) was not found in index patients in other families. Conclusion: The RHOA mutation (R129W) emerges as a potential causative gene for HDGC, but only in one family, indicating a need for further studies to understand its role in HDGC pathogenesis fully.

Targeting the m6A RNA methyltransferase METTL3 attenuates the development of kidney fibrosis.

Kidney fibrosis is a major mechanism underlying chronic kidney disease (CKD). N6-methyladenosine (m6A) RNA methylation is associated with organ fibrosis. We investigated m6A profile alterations and the inhibitory effect of RNA methylation in kidney fibrosis in vitro (TGF-ß-treated HK-2 cells) and in vivo (unilateral ureteral obstruction [UUO] mouse model). METTL3-mediated signaling was inhibited using siRNA in vitro or the METTL3-specific inhibitor STM2457 in vivo and in vitro. In HK-2 cells, METTL3 protein levels increased in a dose- and time-dependent manner along with an increase in the cellular m6A levels. In the UUO model, METTL3 expression and m6A levels were significantly increased. Transcriptomic and m6A profiling demonstrated that epithelial-to-mesenchymal transition- and inflammation-related pathways were significantly associated with RNA m6A methylation. Genetic and pharmacologic inhibition of METTL3 in HK-2 cells decreased TGF-ß-induced fibrotic marker expression. STM2457-induced inhibition of METTL3 attenuated the degree of kidney fibrosis in vivo. Furthermore, METTL3 protein expression was significantly increased in the tissues of CKD patients with diabetic or IgA nephropathy. Therefore, targeting alterations in RNA methylation could be a potential therapeutic strategy for treating kidney fibrosis.

Gut bacteria-derived 3-phenylpropionylglycine mitigates adipocyte differentiation of 3T3-L1 cells by inhibiting adiponectin-PPAR pathway.

BACKGROUND: Gut microbiota provide numerous types of metabolites that humans cannot produce and have a huge influence on the host metabolism. Accordingly, gut bacteria-derived metabolites can be employed as a resource to develop anti-obesity and metabolism-modulating drugs. OBJECTIVE: This study aimed to examine the anti-adipogenic effect of 3-phenylpropionylglycine (PPG), which is a glycine conjugate of bacteria-derived 3-phenylpropionic acid (PPA). METHODS: The effect of PPG on preadipocyte-to-adipocyte differentiation was evaluated in 3T3-L1 differentiation models and the degree of the differentiation was estimated by Oil red O staining. The molecular mechanisms of the PPG effect were investigated with transcriptome analyses using RNA-sequencing and quantitative real-time PCR. RESULTS: PPG suppressed lipid droplet accumulation during the adipogenic differentiation of 3T3-L1 cells, which is attributed to down-regulation of lipogenic genes such as acetyl CoA carboxylase 1 (Acc1) and fatty acid synthase (Fasn). However, other chemicals with chemical structures similar to PPG, including cinnamoylglycine and hippuric acid, had little effect on the lipid accumulation of 3T3-L1 cells. In transcriptomic analysis, PPG suppressed the expression of adipogenesis and metabolism-related gene sets, which is highly associated with downregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Protein-protein association network analysis suggested adiponectin as a hub gene in the network of genes that were differentially expressed genes in response to PPG treatment. CONCLUSION: PPG inhibits preadipocyte-to-adipocyte differentiation by suppressing the adiponectin-PPAR pathway. These data provide a potential candidate from bacteria-derived metabolites with anti-adipogenic effects.

METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m6A modifications and splicing factors.

N6-methyladenosine (m6A) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as m6A methyltransferases (writers), m6A demethylases (erasers), and m6A-binding proteins (readers). Previous studies have suggested that alterations in m6A regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of m6A-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequencing and analyzed alternative splicing patterns in METTL3-knockdown osteosarcoma U2OS cells. We detected 1,803 alternatively spliced genes in METTL3-knockdown cells compared to the controls and found that cell cycle-related genes were enriched in differentially spliced genes. A comparison of the published MeRIP-seq data for METTL14 with our RNA sequencing data revealed that 70-87% of alternatively spliced genes had an m6A peak near 1 kb of alternative splicing sites. Among the 19 RNA-binding proteins enriched in alternative splicing sites, as revealed by motif analysis, expression of SFPQ highly correlated with METTL3 expression in 12,839 TCGA pan-cancer patients. We also found that cell cycle-related genes were enriched in alternatively spliced genes of other cell lines with METTL3 knockdown. Taken together, we suggest that METTL3 regulates m6A-dependent alternative splicing, especially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.

Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation.

Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, kinase, and protein disulfide isomerase (PDI) activities. Of these, transamidase-mediated modification of proteins regulates apoptosis, differentiation, inflammation, and fibrosis. TG2 is highly expressed in mesenchymal stem cells (MSCs) compared with differentiated cells, suggesting a role of TG2 specific for MSC characteristics. In this study, we report a new function of TG2 in the regulation of MSC redox homeostasis. During in vitro MSC expansion, TG2 is required for cell proliferation and self-renewal by preventing premature senescence but has no effect on the expression of surface antigens and oxidative stress-induced cell death. Moreover, induction of differentiation upregulates TG2 that promotes osteoblastic differentiation. Molecular analyses revealed that TG2 mediates tert-butylhydroquinone, but not sulforaphane, -induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in a transamidase activity-independent manner. Differences in the mechanism of action between two NRF2 activators suggest that PDI activity of TG2 may be implicated in the stabilization of NRF2. The role of TG2 in the regulation of antioxidant response was further supported by transcriptomic analysis of MSC. These results indicate that TG2 is a critical enzyme in eliciting antioxidant response in MSC through NRF2 activation, providing a target for optimizing MSC manufacturing processes to prevent premature senescence.

Comparative analysis of cancer gene mutations using targeted sequencing in matched primary and recurrent gastric cancers after chemotherapy.

BACKGROUND: Investigation of responsiveness-associated genes using longitudinal mutation analyses after standard treatments in recurrent gastric cancer (GC) is limited. OBJECTIVE: To evaluate the somatic mutations associated with resistance to combined treatment involving fluorouracil (FU) or platinum (PL) in advanced GC. METHODS: Samples from patients with advanced GC treated with FU or PL alone, or surgery plus FU/PL, were studied. GC patients who relapsed after standard chemotherapy (FU/PL) and with presence of tumor samples from initial diagnosis and recurrence were included. Targeted sequencing analysis of 143 cancer-related genes was performed using an Oncomine Comprehensive Cancer Panel. RESULTS: Matched samples of primary and recurrent lesions were analyzed in sixteen patients with GC. When genes with recurrent mutations in two or more patients were used as specific findings, a total of 26 genes were found. TP53 was the most predominantly increased allele frequency (AF) in recurrent GCs after standard treatment. The mutational AF of ERBB2, PTEN, and BRCA2 also commonly increased, suggesting the role of these mutations in treatment resistance, whereas the mutational AF of VLH, NF1, and STK11 frequently decreased in recurrent tumors, suggesting the role of these mutations in increasing sensitivity to treatment. TCGA gastric cancer data (n = 436) were analyzed, and mutation sites detected in 16 GC patients in this study were in agreement with TCGA cohort with some exceptions. Overall survival according to gene expression associated with chemotherapy responsiveness exhibited compatible patterns with gain or loss-of-function mutations of each gene. CONCLUSIONS: Mutations in TP53, ERBB2, PTEN, BRCA2, VHL, NF1, and STK11 are candidate somatic alterations related to chemoresistance in GC.

Polo-Like Kinase 1 Regulates Chromosomal Instability and Paclitaxel Resistance in Breast Cancer Cells.

PURPOSE: Chromosomal instability (CIN) contributes to intercellular genetic heterogeneity and has been implicated in paclitaxel (PTX) resistance in breast cancer. In this study, we explored polo-like kinase 1 (PLK1) as an important regulator of mitotic integrity and as a useful predictive biomarker for PTX resistance in breast cancer. METHODS: We performed PTX resistance screening using the human kinome CRISPR/Cas9 library in breast cancer cells. In vitro cell proliferation and apoptosis assays and in vivo xenograft experiments were performed to determine the effects of PLK1 on breast cancer cells. Immunofluorescence microscopy was used to measure the degree of multipolar cell division. RESULTS: Kinome-wide CRISPR/Cas9 screening identified various kinases involved in PTX resistance in breast cancer cells; among these, PLK1 was chosen for further experiments. PLK1 knockdown inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro and in vivo. Moreover, PLK1 silencing sensitized breast cancer cells and mouse xenograft tumor models to PTX cytotoxicity. Silencing of PLK1 induced the formation of multipolar spindles and increased the percentage of multipolar cells. In addition, PLK1 silencing resulted in the downregulation of BubR1 and Mad2 in breast cancer cells. Furthermore, PLK1 upregulation in primary breast cancer was associated with decreased overall patient survival based on the analysis of The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium databases. CONCLUSION: PLK1 plays an important role in PTX resistance by regulating CIN in breast cancer cells. Targeting PLK1 may be an effective treatment strategy for PTX-resistant breast cancers.

Augmentation of the RNA m6A reader signature is associated with poor survival by enhancing cell proliferation and EMT across cancer types.

N6-Methyladenosine (m6A) RNA modification plays a critical role in the posttranscriptional regulation of gene expression. Alterations in cellular m6A levels and m6A-related genes have been reported in many cancers, but whether they play oncogenic or tumor-suppressive roles is inconsistent across cancer types. We investigated common features of alterations in m6A modification and m6A-related genes during carcinogenesis by analyzing transcriptome data of 11 solid tumors from The Cancer Genome Atlas database and our in-house gastric cancer cohort. We calculated m6A writer (W), eraser (E), and reader (R) signatures based on corresponding gene expression. Alterations in the W and E signatures varied according to the cancer type, with a strong positive correlation between the W and E signatures in all types. When the patients were divided according to m6A levels estimated by the ratio of the W and E signatures, the prognostic effect of m6A was inconsistent according to the cancer type. The R and especially the R2 signatures (based on the expression of IGF2BPs) were upregulated in all cancers. Patients with a high R2 signature exhibited poor prognosis across types, which was attributed to enrichment of cell cycle- and epithelial-mesenchymal transition-related pathways. Our study demonstrates common features of m6A alterations across cancer types and suggests that targeting m6A R proteins is a promising strategy for cancer treatment.

Direct cell-to-cell transfer in stressed tumor microenvironment aggravates tumorigenic or metastatic potential in pancreatic cancer.

Pancreatic cancer exhibits a characteristic tumor microenvironment (TME) due to enhanced fibrosis and hypoxia and is particularly resistant to conventional chemotherapy. However, the molecular mechanisms underlying TME-associated treatment resistance in pancreatic cancer are not fully understood. Here, we developed an in vitro TME mimic system comprising pancreatic cancer cells, fibroblasts and immune cells, and a stress condition, including hypoxia and gemcitabine. Cells with high viability under stress showed evidence of increased direct cell-to-cell transfer of biomolecules. The resulting derivative cells (CD44high/SLC16A1high) were similar to cancer stem cell-like-cells (CSCs) with enhanced anchorage-independent growth or invasiveness and acquired metabolic reprogramming. Furthermore, CD24 was a determinant for transition between the tumorsphere formation or invasive properties. Pancreatic cancer patients with CD44low/SLC16A1low expression exhibited better prognoses compared to other groups. Our results suggest that crosstalk via direct cell-to-cell transfer of cellular components foster chemotherapy-induced tumor evolution and that targeting of CD44 and MCT1(encoded by SLC16A1) may be useful strategy to prevent recurrence of gemcitabine-exposed pancreatic cancers.

Transglutaminase 2 mediates transcriptional regulation through BAF250a polyamination.

BACKGROUND: Transglutaminase 2 (TG2) mediates protein modifications by crosslinking or by incorporating polyamine in response to oxidative or DNA-damaging stress, thereby regulating apoptosis, extracellular matrix formation, and inflammation. The regulation of transcriptional activity by TG2-mediated histone serotonylation or by Sp1 crosslinking may also contribute to cellular stress responses. OBJECTIVE: In this study, we attempted to identify TG2-interacting proteins to better understand the role of TG2 in transcriptional regulation. METHODS: Using a yeast two-hybrid assay to screen a HeLa cell cDNA library, we found that TG2 bound BAF250a, a core subunit of the cBAF chromatin remodeling complex, through an interaction between the TG2 barrel 1 and BAF250a C-terminal domains. RESULTS: TG2 was pulled down with a GST-BAF250a C-term fusion protein. Moreover, TG2 and BAF250a were co-fractionated using P11 chromatography, and co-immunoprecipitated. A transamidation reaction showed that TG2 mediated incorporation of polyamine into BAF250a. In glucocorticoid response-element reporter-expressing cells, TG2 overexpression increased the luciferase reporter activity in a transamidation-dependent manner. In addition, a comparison of genome-wide gene expression between wild-type and TG2-deficient primary hepatocytes in response to dexamethasone treatment showed that TG2 further enhanced or suppressed the expression of dexamethasone-regulated genes that were identified by a gene ontology enrichment analysis. CONCLUSION: Thus, our results indicate that TG2 regulates transcriptional activity through BAF250a polyamination.

Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein-protein interactions of the fused protein.

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein-protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein-protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.