Integrated Analysis of Microbiome and Metabolome Reveals Disease-Specific Profiles in Inflammatory Bowel Diseases and Intestinal Behçet's Disease.

The gut microbial and metabolic characteristics of intestinal Behçet's disease (BD), a condition sharing many clinical similarities with ulcerative colitis (UC) and Crohn's disease (CD), are largely unexplored. This study investigated the gut microbial and metabolic characteristics of intestinal BD as well as potential biomarkers, comparing them with those in UC, CD, and healthy controls. Colon tissue and stool samples from 100 patients (35 UC, 30 CD, and 35 intestinal BD) and 41 healthy volunteers were analyzed using 16S ribosomal RNA sequencing to assess microbial diversity, taxonomic composition, and functional profiling. Plasma metabolomic analyses were performed using gas chromatography and ultra-performance liquid chromatography-mass spectrometry. Results indicated reduced microbial diversity in CD but not in intestinal BD, with intestinal BD showing fewer changes compared to controls yet distinct taxonomic features from UC, CD, and controls. Common alterations across all diseases included a reduction in beneficial bacteria producing short-chain fatty acids. Intestinal BD-specific changes featured a decreased abundance of Bacteroides fragilis. Metabolomic profiles in intestinal BD were similar to those in CD but distinct from those in UC, displaying significant changes in energy metabolism and genetic information processing. This integrative analysis revealed both shared and unique profiles in intestinal BD compared with UC, CD, and controls, advancing our understanding of the distinctive features of these diseases.

New genetic biomarkers predicting 5-aminosalicylate-induced adverse events in patients with inflammatory bowel diseases.

Background: Notably, 5-aminosalicylates (5-ASA) are vital in treating inflammatory bowel diseases (IBD). The adverse events of 5-ASA rarely occur but they could be fatal. Objectives: We aimed to discover new genetic biomarkers predicting 5-ASA-induced adverse events in patients with IBD. Design: This was a retrospective observational study. Methods: We performed a genome-wide association study on patients with IBD in South Korea. We defined subset 1 as 39 all adverse events and 272 controls; subset 2 as 20 severe adverse events and 291 controls (mild adverse events and control); subset 3 as 20 severe adverse events and 272 controls; and subset 4 as 19 mild adverse events and 272 controls. Logistic regression analysis was performed and commonly found associated genes were determined as candidate single-nucleotide polymorphisms predicting 5-ASA adverse events. Results: Patients with Crohn's disease (CD) were significantly negatively associated with the development of adverse events compared to patients with ulcerative colitis (UC) (5.3% versus 22.9%). However, sex and age at diagnosis were unassociated with the adverse events of 5-ASA. rs13898676 [odds ratio (OR), 20.33; 95% confidence interval (CI), 5.69-72.67; p = 3.57 × e-6], rs12681590 (OR, 7.35; 95% CI, 2.85-19.00; p = 3.78 × e-5), rs10967320 (OR, 4.51; 95% CI, 2.18-9.31; p = 4.72 × e-5), and rs78726924 (OR, 3.54; 95% CI, 1.69-7.40; p = 7.96 × e-5) were genetic biomarkers predicting 5-ASA-induced severe adverse events in patients with IBD. Conclusion: The adverse events of 5-ASA were more common in patients with UC than those with CD in our study. We found that novel rs13898676 nearby WSB2 was the most significant genetic locus contributing to 5-ASA's adverse event risk.

Bifidobacterium breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

BACKGROUND AND AIM: Bifidobacterium breve was the first bacteria isolated in the feces of healthy infants and is a dominant species in the guts of breast-fed infants. Some strains of B. breve have been shown to be effective at relieving intestinal inflammation, but the modes of action have yet to be elucidated. In this study, we investigated the mechanisms of action of B. breve CBT BR3 isolated from South Korean infant feces in relieving colitis in vitro and in vivo. METHODS: Colitis was induced in mice with dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS). Quantitative reverse-transcription polymerase chain reaction, in vitro FITC-dextran flux permeability assay, and aryl hydrocarbon receptor (AhR) luciferase assay are performed using Caco-2 cells and HT29-Lucia™ AhR cells. RESULTS: B. breve CBT BR3 was orally administered. B. breve CBT BR3 improved colitis symptoms in both DSS- and DNBS-induced colitis models. B. breve CBT BR3 increased the number of goblet cells per crypt. B. breve increased the mRNA expressions of Notch, Spdef, Muc5, and Il22. The mRNA expressions of Occludin, which encodes a membrane tight-junction protein, and Foxo3, which encodes a protein related to butyrate metabolism, were also increased in the DSS- and DNBS-induced colitis models. B. breve CBT BR3 protected inflammation-induced epithelial cell permeability and improved goblet cell function by inducing aryl hydrocarbon receptor in vitro. CONCLUSIONS: These results indicate that B. breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

Interplay between chronic inflammation and clonal haematopoiesis of indeterminate potential in Behçet's disease.

BACKGROUND: Clonal haematopoiesis of indeterminate potential (CHIP) is a predisposition to haematological malignancy whose relationship with chronic inflammatory diseases, such as cardiovascular diseases, has been highlighted. Here, we aimed to investigate the CHIP emergence rate and its association with inflammatory markers in Behçet's disease (BD). METHODS: We performed targeted next-generation sequencing to detect the presence of CHIP using peripheral blood cells from 117 BD patients and 5004 healthy controls between March 2009 and September 2021 and analysed the association between CHIP and inflammatory markers. RESULTS: CHIP was detected in 13.9% of patients in the control group and 11.1% of patients in the BD group, indicating no significant intergroup difference. Among the BD patients of our cohort, five variants (DNMT3A, TET2, ASXL1, STAG2, and IDH2) were detected. DNMT3A mutations were the most common, followed by TET2 mutations. CHIP carriers with BD had a higher serum platelet count, erythrocyte sedimentation rate, and C-reactive protein level; older age; and lower serum albumin level at diagnosis than non-CHIP carriers with BD. However, the significant association between inflammatory markers and CHIP disappeared after the adjustment for various variables, including age. Moreover, CHIP was not an independent risk factor for poor clinical outcomes in patients with BD. CONCLUSIONS: Although BD patients did not have higher CHIP emergence rates than the general population, older age and degree of inflammation in BD were associated with CHIP emergence.

Downregulation of Heat Shock Protein 72 Contributes to Fibrostenosis in Crohn's Disease.

Background/Aims: Crohn's disease (CD) with recurrent inflammation can cause intestinal fibrostenosis due to dysregulated deposition of extracellular matrix. However, little is known about the pathogenesis of fibrostenosis. Here, we performed a differential proteomic analysis between normal, inflamed, and fibrostenotic specimens of patients with CD and investigated the roles of the candidate proteins in myofibroblast activation and fibrosis. Methods: We performed two-dimensional difference gel electrophoresis and identified candidate proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and orbitrap liquid chromatography-mass spectrometry. We also verified the levels of candidate proteins in clinical specimens and examined their effects on 18Co myofibroblasts and Caco-2 intestinal epithelial cells. Results: We identified five of 30 proteins (HSP72, HSPA5, KRT8, PEPCK-M, and FABP6) differentially expressed in fibrostenotic CD. Among these proteins, the knockdown of heat shock protein 72 (HSP72) promoted the activation and wound healing of myofibroblasts. Moreover, knockdown of HSP72 induced the epithelial-mesenchymal transition of intestinal epithelial cells by reducing E-cadherin and inducing fibronectin and α-smooth muscle actin, which contribute to fibrosis. Conclusions: HSP72 is an important mediator that regulates myofibroblasts and epithelial-mesenchymal transition in fibrosis of CD, suggesting that HSP72 can serve as a target for antifibrotic therapy.

Olfactomedin 4 produces dysplasia but suppresses metastasis of colon cancer.

Development of colorectal cancer (CRC) is regulated by a series of genetic and microenvironmental alterations. Olfactomedin 4 (OLFM4) is a secreted glycoprotein that is highly expressed in the gastrointestinal tract and modulates inflammation. However, the role of OLFM4 in CRC is uncertain. Here we aimed to explore the function of OLFM4 in CRC in vivo and in vitro. The mRNA expression of OLFM4 was up-regulated in precursor lesions with dysplasia or ulcerative colitis but was reduced in CRC. OLFM4 neutralizing antibody suppressed inflammation-mediated early-stage CRC formation in an AOM/DSS colitis-associated cancer model. OLFM4 knockdown cells exhibited increased cell proliferation and motility in vitro and in vivo. Ablation of OLFM4 increased tumor growth and metastasis in xenograft experiments. In addition, OLFM4 knockdown cells showed elevated expression of colon cancer stem cell markers including CD133, resulting in increased metastasis via epithelial-mesenchymal transition signaling. This study demonstrated that OLFM4 regulates inflammation and cancer progression differently; ablation of OLFM4 promotes cancer metastasis via stemness and epithelial-mesenchymal transition. These results suggest a new route for controlling cancer progression and metastasis.

Novel Histone Deacetylase 6 Inhibitor Confers Anti-inflammatory Effects and Enhances Gut Barrier Function.

Background/Aims: The purpose of the current study was to examine the anti-inflammatory effects of CKD-506, a novel histone deacetylase 6 inhibitor, on human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and to explore the relationship between CKD-506 and gut epithelial barrier function. Methods: Lipopolysaccharide-stimulated human PBMCs from inflammatory bowel disease (IBD) patients were treated with CKD-506, and tumor necrosis factor (TNF)-α expression was measured using an enzyme-linked immunosorbent assay. The proliferation of CD4+ T cells from IBD patients was evaluated using flow cytometric analysis. The effects of CKD-506 on gut barrier function in a cell line and colon organoids, based on examinations of mRNA production, goblet cell differentiation, and E-cadherin recovery, were investigated using quantitative reverse transcription polymerase chain reaction, immunofluorescence, and a fluorescein isothiocyanate-dextran permeability assay. Results: Secretion of TNF-α, a pivotal pro-inflammatory mediator in IBD, by lipopolysaccharide-triggered PBMCs was markedly decreased by CKD-506 treatment in a dose-dependent manner and to a greater extent than by tofacitinib or tubastatin A treatment. E-cadherin mRNA expression and goblet cell differentiation increased significantly and dose-dependently in HT-29 cells in response to CKD-506, and inhibition of E-cadherin loss after TNF-α stimulation was significantly reduced both in HT-29 cells and gut organoids. Caco-2 cells treated with CKD-506 showed a significant reduction in barrier permeability in a dose-dependent manner. Conclusions: The present study demonstrated that CKD-506 has anti-inflammatory effects on PBMCs and CD4 T cells and improves gut barrier function, suggesting its potential as a small-molecule therapeutic option for IBD.

Novel Potassium-Competitive Acid Blocker, Tegoprazan, Protects Against Colitis by Improving Gut Barrier Function.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder characterized by prolonged inflammation of the gastrointestinal tract. IBD can result from gut barrier dysfunction, altered gut microbiota, and abnormal intestinal immunity induced by environmental factors in genetically susceptible individuals. Proton pump inhibitors (PPIs) such as rabeprazole are frequently employed for gastric acid inhibition. However, long-term PPI administration can alter the intestinal microbiome composition, possibly worsening IBD severity. The present study revealed that tegoprazan, a potassium-competitive acid blocker, significantly improved colitis in mice and enhanced the intestinal epithelial barrier function. Tegoprazan alleviated gut microbiota dysbiosis and enhanced the growth of Bacteroides vulgatus. In turn, B. vulgatus alleviated intestinal inflammation by inhibiting epithelial adhesion of pathogenic bacteria. Unlike rabeprazole, tegoprazan did not induce gut dysbiosis. Our findings provide novel insights into the potential role of tegoprazan as an intestinal protectant for IBD and as a therapeutic agent for gastric acid-related diseases.

A GLP-1/GLP-2 receptor dual agonist to treat NASH: Targeting the gut-liver axis and microbiome.

BACKGROUND AND AIMS: Currently there is no Food and Drug Administration-approved drug to treat NAFLD and NASH, the rates of which are increasing worldwide. Although NAFLD/NASH are highly complex and heterogeneous conditions, most pharmacotherapy pipelines focus on a single mechanistic target. Considering the importance of the gut-liver axis in their pathogenesis, we investigated the therapeutic effect of a long-acting dual agonist of glucagon-like peptide (GLP)-1 and GLP-2 receptors in mice with NAFLD/NASH. APPROACH AND RESULTS: C57BL/6J mice were fed a choline-deficient high-fat diet/high fructose and sucrose solution. After 16 weeks, mice were randomly allocated to receive vehicle, GLP1-Fc, GLP2-Fc, or GLP1/2-Fc fusion (GLP1/2-Fc) subcutaneously every 2 days for 4 weeks. Body weight was monitored, insulin/glucose tolerance tests were performed, feces were collected, and microbiome profiles were analyzed. Immobilized cell systems were used to evaluate direct peptide effect. Immunohistochemistry, quantitative PCR, immunoblot analysis, tunnel assay, and biochemical assays were performed to assess drug effects on inflammation, hepatic fibrosis, cell death, and intestinal structures. The mice had well-developed NASH phenotypes. GLP1/2-Fc reduced body weight, glucose levels, hepatic triglyceride levels, and cellular apoptosis. It improved liver fibrosis, insulin sensitivity, and intestinal tight junctions, and increased microvillus height, crypt depth, and goblet cells of intestine compared with a vehicle group. Similar effects of GLP1/2-Fc were found in in vitro cell systems. GLP1/2-Fc also changed microbiome profiles. We applied fecal microbiota transplantation (FMT) gain further insight into the mechanism of GLP1/2-Fc-mediated protection. We confirmed that FMT exerted an additive effect on GLP1-Fc group, including the body weight change, liver weight, hepatic fat accumulation, inflammation, and hepatic fibrosis. CONCLUSIONS: A long-acting dual agonist of GLP-1 and GLP-2 receptors is a promising therapeutic strategy to treat NAFLD/NASH.

A transepithelial pathway delivers succinate to macrophages, thus perpetuating their pro-inflammatory metabolic state.

The gut metabolite composition determined by the microbiota has paramount impact on gastrointestinal physiology. However, the role that bacterial metabolites play in communicating with host cells during inflammatory diseases is poorly understood. Here, we aim to identify the microbiota-determined output of the pro-inflammatory metabolite, succinate, and to elucidate the pathways that control transepithelial succinate absorption and subsequent succinate delivery to macrophages. We show a significant increase of succinate uptake into pro-inflammatory macrophages, which is controlled by Na+-dependent succinate transporters in macrophages and epithelial cells. Furthermore, we find that fecal and serum succinate concentrations were markedly augmented in inflammatory bowel diseases (IBDs) and corresponded to changes in succinate-metabolizing gut bacteria. Together, our results describe a succinate production and transport pathway that controls the absorption of succinate generated by distinct gut bacteria and its delivery into macrophages. In IBD, this mechanism fails to protect against the succinate surge, which may result in chronic inflammation.

An Escherichia coli strain with extra catalase activity protects against murine colitis by scavenging hydrogen peroxide and regulating regulatory t cell/interleukin-17 pathways.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract whose occurrence is attributed to various factors, including genetic factors, immune response, microbial changes, and oxidative stress. Microbial-targeted therapy has emerged as an alternative to immunosuppressive therapy for IBD. METHODS: The effects of an atypical commensal Escherichia coli strain harboring an additional catalase gene (compared to typical E. coli strain) on dextran sulfate sodium (DSS)-induced colitis were explored in mice. RESULTS: The atypical E. coli (atEc) significantly restored body weight, reduced disease activity score, and improved histological scores in mice with colitis. Hydrogen peroxide levels in colitis mice were noticeably decreased when the mice were administered atEc. The proinflammatory cytokine levels were decreased and regulatory T cell numbers were increased after the administration of atEc. The abundance of Firmicutes was significantly recovered, while that of Proteobacteria decreased in atEc -treated mice compared with that in vehicle-treated wild-type mice. To investigate the role of interleukin (IL)-17A in mediating the anti-inflammatory effects of the atEc, IL-17A‒knockout mice were orally administered atEc. Clinical and immune responses and microbial composition were significantly reduced in IL-17A‒knockout mice compared with those in wild-type mice. CONCLUSIONS: atEc ameliorates colonic inflammation by controlling hydrogen peroxide levels, immune responses (including regulatory T cells and IL-17A), and microbial composition. atEc could be a novel candidate of probiotic for IBD treatment.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Argininosuccinate synthase 1 suppresses tumor progression through activation of PERK/eIF2α/ATF4/CHOP axis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide, and liver cancer has increased in mortality due to liver cancer because it was detected at an advanced stages in patients with liver dysfunction, making HCC a lethal cancer. Accordingly, we aim to new targets for HCC drug discovery using HCC tumor spheroids. METHODS: Our comparative proteomic analysis of HCC cells grown in culture as monolayers (2D) and spheroids (3D) revealed that argininosuccinate synthase 1 (ASS1) expression was higher in 3D cells than in 2D cells due to upregulated endoplasmic reticulum (ER) stress responses. We investigated the clinical value of ASS1 in Korean patients with HCC. The mechanism underlying ASS1-mediated tumor suppression was investigated in HCC spheroids. ASS1-mediated improvement of chemotherapy efficiency was observed using high content screening in an HCC xenograft mouse model. RESULTS: Studies of tumor tissue from Korean HCC patients showed that, although ASS1 expression was low in most samples, high levels of ASS1 were associated with favorable overall survival of patients. Here, we found that bidirectional interactions between ASS1 ER stress responses in HCC-derived multicellular tumor spheroids can limit HCC progression. ASS1 overexpression effectively inhibited tumor growth and enhanced the efficacy of in vitro and in vivo anti-HCC combination chemotherapy via activation of the PERK/eIF2α/ATF4/CHOP axis, but was not dependent on the status of p53 and arginine metabolism. CONCLUSIONS: These results demonstrate the critical functional roles for the arginine metabolism-independent tumor suppressor activity of ASS1 in HCC and suggest that upregulating ASS1 in these tumors is a potential strategy in HCC cells with low ASS1 expression.

Succinate-treated macrophages attenuate dextran sodium sulfate colitis in mice.

BACKGROUND/AIMS: The safety and effectiveness of adalimumab was demonstrated in a phase 3 trial in Japanese patients with intestinal Behçet's disease. The aim of this study was to evaluate the long-term safety and effectiveness of adalimumab in Japanese patients with intestinal Behçet's disease.

Nanocomposites-based targeted oral drug delivery systems with infliximab in a murine colitis model.

BACKGROUND: Infliximab (IFX), a TNF-α blocking chimeric monoclonal antibody, induces clinical response and mucosal healing in patients with inflammatory bowel disease (IBD). However, systemic administration of this agent causes unwanted side effects. Oral delivery of antibody therapeutics might be an effective treatment strategy for IBD compared to intravenous administration. RESULTS: All three carriers had a high encapsulation efficiency, narrow size distribution, and minimal systemic exposure. There was a higher interaction between nanocomposite carriers and monocytes compared to lymphocytes in the PBMC of IBD patients. Orally administered nanocomposite carriers targeted to inflamed colitis minimized systemic exposure. All IFX delivery formulations with nanocomposite carriers had a significantly less colitis-induced body weight loss, colon shortening and histomorphological score, compared to the DSS-treated group. AC-IFX-L and EAC-IFX-L groups showed significantly higher improvement of the disease activity index, compared to the DSS-treated group. In addition, AC-IFX-L and EAC-IFX-L alleviated pro-inflammatory cytokine expressions (Tnfa, Il1b, and Il17). CONCLUSION: We present orally administered antibody delivery systems which improved efficacy in murine colitis while reducing systemic exposure. These oral delivery systems suggest a promising therapeutic approach for treating IBD.

Identification of inhibitors of Bcl-2 family protein-protein interaction by combining the BRET screening platform with virtual screening.

Bcl-2 family proteins play key roles in tumor initiation, progression, and resistance to therapy. Therefore, the protein-protein interactions (PPIs) between the pro-survival proteins, B-cell lymphoma (Bcl)-2 and Bcl-xL, and the pro-apoptotic proteins, Bax and Bak, could be attractive therapeutic targets for anti-cancer drug discovery. Here, we found new small molecules, BIP-A1001 and BIP-A2001 that modulated Bak/Bax and Bcl-xL interactions by combining the Nanoluc/YFP-based bioluminescence resonance energy transfer (BRET) assay with structure based virtual screening. In addition, we chose compounds with similar structures to BIP-A1001 and BIP-A2001 and tested their inhibitory effects using the BRET assay as a dose-response function. The results indicated that identifying compounds that inhibit interactions between Bak/Bax and Bcl-xL could be a promising approach to enhance cancer therapy.

Actinomycin D inhibits the expression of the cystine/glutamate transporter xCT via attenuation of CD133 synthesis in CD133+ HCC.

Liver cancer is one of the most frequently occurring types of cancer with high mortality rate. Hepatocellular carcinoma (HCC) frequently metastasizes to lung, portal vein, and portal lymph nodes and most HCCs show strong resistance to conventional anticancer drugs. Cancer stem cells (CSCs) are considered to be responsible for resistance to therapies. Hence, recent advancements in the use of liver cancer stem cells (LCSCs) are rapidly gaining recognition as an efficient and organized means for developing antitumor agents. We aimed to use a non-target-based high-throughput screening (HTS) approach to specifically target α-fetoprotein (AFP)+/cluster of differentiation (CD)133+ HCC present in mixed populations of HCC cells and hepatocytes. Herein, we identified actinomycin D (ActD) as a potential antitumor agent that significantly inhibits activity of LCSCs without affecting the co-cultured hepatocytes. To determine the mechanism of ActD-induced tumor-specificity in LCSC, we applied various cell-based assay models in vitro. In fact, ActD significantly increased reactive oxygen species (ROS) accumulation and DNA damage in Huh7 HCC cells, but not in Fa2N-4 cells, immortalized hepatocytes. Treatment of spheroid-forming LCSCs with ActD effectively decreased spheroid formation and the CD133+ HCC cell population. Importantly, these ActD-mediated effects are a result of inhibition of cystine/glutamate transporter xCT expression, via attenuation of CD133 synthesis. These results indicate that ActD suppresses stemness and malignant properties in HCC cells through destabilization of xCT, by inhibition of CD133 expression in LCSCs. The effects of ActD on LCSCs provide novel therapeutic strategies for targeting cancer stem-like cells in liver cancer.

SUMOylation regulates nuclear localization and stability of TRAIP/RNF206.

TRAIP/RNF206 plays diverse roles in cell cycle progression, DNA damage response, and DNA repair pathways. Physiological importance of TRAIP is highlighted by the identification of pathogenic mutations of TRAIP gene in patients diagnosed with primordial dwarfism. Although the diverse functions of TRAIP in the nucleus have been well characterized, molecular mechanism of TRAIP retention in the nucleus has not been determined. Here, we discovered that TRAIP is post-translationally modified by the small ubiquitin-like protein (SUMO). In addition, we identified five SUMOylation sites in TRAIP, and successfully generated SUMOylation deficient mutant of TRAIP. In an attempt to define the functional roles of TRAIP SUMOylation, we discovered that SUMOylation deficient TRAIP is not retained in the nucleus. In addition, protein stability of SUMOylation deficient TRAIP is lower than wild type TRAIP, demonstrating that SUMOylation is critical for both proper subcellular localization and protein stability of TRAIP. Taken together, these findings improve the understanding clinical implication of TRAIP in various diseases including primordial dwarfism and cancers.