Supplementation of nicotinamide mononucleotide diminishes COX-2 associated inflammatory responses in macrophages by activating kynurenine/AhR signaling.

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for prostaglandin synthesis during inflammation and immune responses. Our previous results show that NAD+ level decreased in activated macrophages while nicotinamide mononucleotide (NMN) supplementation suppressed the inflammatory responses via restoring NAD+ level and downregulating COX-2. However, whether NMN downregulates COX-2 in mouse model of inflammation, and its underlying mechanism needs to be further explored. In the present study, we established LPS- and alum-induced inflammation model and demonstrated that NMN suppressed the inflammatory responses in vivo. Quantitative proteomics in mouse peritoneal macrophages identified that NMN activated AhR signaling pathway in activated macrophages. Furthermore, we revealed that NMN supplementation led to IDO1 activation and kynurenine accumulation, which caused AhR nuclear translocation and activation. On the other hand, AhR or IDO1 knockout abolished the effects of NMN on suppressing COX-2 expression and inflammatory responses in macrophages. In summary, our results demonstrated that NMN suppresses inflammatory responses by activating IDO-kynurenine-AhR pathway, and suggested that administration of NMN in early-stage immuno-activation may cause an adverse health effect.

HER2 phosphorylation induced by TGF-ß promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor ß (TGF-ß) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-ß can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-ß enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-ß type I receptor TßRI kinase activity. TßRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-ß-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-ß-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-ß in breast cancer progression.

Flavonoid 4,4'-dimethoxychalcone selectively eliminates senescent cells via activating ferritinophagy.

Flavonoids are bioactive natural polyphenolic compounds with health benefits, including anti-tumor, anti-inflammatory and anti-aging effects. Our previous studies revealed that a flavonoid 4,4'-dimethoxychalcone (DMC) induced ferroptosis via inhibiting ferrochelatase (FECH). However, the effect of DMC on cellular senescence is unknown. In the present study, we found that DMC treatment selectively eliminated senescent cells, and DMC alone or a combination of DMC and quercetin or dasatinib showed high efficiency in the clearance of senescent cells. We identified FECH was highly expressed in senescent cells compared to non-senescent cells. Mechanistically, we found that DMC inhibited FECH and induced ferritinophagy, which led to an increase of labile iron pool, triggering ferroptosis of senescent cells. Importantly, we found that DMC treatment prevented hair loss, improved motor coordination, and reduced the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1ß, CXCL-10, and MMP12) in the liver of old mice. Collectively, we revealed that, through the induction of ferroptosis, DMC holds the promise as a new senolytics to prevent age-related pathologies.

Age-related noncanonical TRMT6-TRMT61A signaling impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity and differentiation bias toward myeloid lineages. However, the molecular mechanism behind HSC aging remains largely unknown. In this study, we observed that RNA N1-methyladenosine-generating methyltransferase TRMT6-TRMT61A complex is increased in aged murine HSCs due to aging-declined CRL4DCAF1-mediated ubiquitination degradation signaling. Unexpectedly, no difference of tRNA N1-methyladenosine methylome is observed between young and aged hematopoietic stem and progenitor cells, suggesting a noncanonical role of the TRMT6-TRMT61A complex in the HSC aging process. Further investigation revealed that enforced TRMT6-TRMT61A impairs HSCs through 3'-tiRNA-Leu-CAG and subsequent RIPK1-RIPK3-MLKL-mediated necroptosis cascade. Deficiency of necroptosis ameliorates the self-renewal capacity of HSCs and counters the physiologically deleterious effect of enforced TRMT6-TRMT61A on HSCs. Together, our work uncovers a nonclassical role for the TRMT6-TRMT61A complex in HSC aging and highlights a therapeutic target.

Multi-Omics Analyses Reveal Mitochondrial Dysfunction Contributing to Temozolomide Resistance in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor with poor prognosis. Temozolomide (TMZ) is the standard chemotherapy for glioblastoma treatment, but TMZ resistance significantly compromises its efficacy. In the present study, we generated a TMZ-resistant cell line and identified that mitochondrial dysfunction was a novel factor contributing to TMZ resistance though multi-omics analyses and energy metabolism analysis. Furthermore, we found that rotenone treatment induced TMZ resistance to a certain level in glioblastoma cells. Notably, we further demonstrated that elevated Ca2+ levels and JNK-STAT3 pathway activation contributed to TMZ resistance and that inhibiting JNK or STAT3 increases susceptibility to TMZ. Taken together, our results indicate that co-administering TMZ with a JNK or STAT3 inhibitor holds promise as a potentially effective treatment for glioblastoma.

Molecular basis of methyl-salicylate-mediated plant airborne defence.

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2-5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylic acid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylic acid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2-SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid-virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.

Development of pseudo-targeted profiling of isotopic metabolomics using combined platform of high resolution mass spectrometry and triple quadrupole mass spectrometry with application of 13C6-glucose tracing in HepG2 cells.

Isotope tracing assisted metabolic analysis is becoming a unique tool to understand metabolic regulation in cell biology and biomedical research. Targeted mass spectrometry analysis based on selected reaction monitoring (SRM) has been widely applied in isotope tracing experiment with the advantages of high sensitivity and broad linearity. However, its application for new pathway discovery is largely restrained by molecular coverage. To overcome this limitation, we describe a strategy called pseudo-targeted profiling of isotopic metabolomics (PtPIM) to expand the analysis of isotope labeled metabolites beyond the limit of known pathways and chemical standards. Pseudo-targeted metabolomics was first established with ion transitions and retention times transformed from high resolution (orbitrap) mass spectrometry. Isotope labeled MRM transitions were then generated according to chemical formulas of fragments, which were derived from accurate ion masses acquired by HRMS. An in-house software "PseudoIsoMRM" was developed to simulate isotope labeled ion transitions in batch mode and correct the interference of natural isotopologues. This PtPIM strategy was successfully applied to study 13C6-glucose traced HepG2 cells. As 313 molecules determined as analysis targets, a total of 4104 ion transitions were simulated to monitor 13C labeled metabolites in positive-negative switching mode of QQQ mass spectrometer with minimum dwell time of 0.3 ms achieved. A total of 68 metabolites covering glycolysis, TCA cycle, nucleotide biosynthesis, one-carbon metabolism and related derivatives were found to be labeled (> 2%) in HepG2 cells. Active pentose phosphate pathway was observed with diverse labeling status of glycolysis intermediates. Meanwhile, our PtPIM strategy revealed that rotenone severely suppressed mitochondrial function e.g. oxidative phosphorylation and fatty acid beta-oxidation. In this case, anaerobic respiration became the major source of energy metabolism by producing abundant lactate. Conclusively, the simulation based PtPIM method demonstrates a strategy to broaden metabolite coverage in isotope tracing analysis independent of standard chemicals.

PRMT5 methylating SMAD4 activates TGF-ß signaling and promotes colorectal cancer metastasis.

Perturbations in transforming growth factor-ß (TGF-ß) signaling can lead to a plethora of diseases, including cancer. Mutations and posttranslational modifications (PTMs) of the partner of SMAD complexes contribute to the dysregulation of TGF-ß signaling. Here, we reported a PTM of SMAD4, R361 methylation, that was critical for SMAD complexes formation and TGF-ß signaling activation. Through mass spectrometric, co-immunoprecipitation (Co-IP) and immunofluorescent (IF) assays, we found that oncogene protein arginine methyltransferase 5 (PRMT5) interacted with SMAD4 under TGF-ß1 treatment. Mechanically, PRMT5 triggered SMAD4 methylation at R361 and induced SMAD complexes formation and nuclear import. Furthermore, we emphasized that PRMT5 interacting and methylating SMAD4 was required for TGF-ß1-induced epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis, and SMAD4 R361 mutation diminished PRMT5 and TGF-ß1-induced metastasis. In addition, highly expressed PRMT5 or high level of SMAD4 R361 methylation indicated worse outcomes in clinical specimens analysis. Collectively, our study highlights the critical interaction of PRMT5 and SMAD4 and the roles of SMAD4 R361 methylation for controlling TGF-ß signaling during metastasis. We provided a new insight for SMAD4 activation. And this study indicated that blocking PRMT5-SMAD4 signaling might be an effective targeting strategy in SMAD4 wild-type CRC.

Bcl10 phosphorylation-dependent droplet-like condensation positively regulates DNA virus-induced innate immune signaling.

B-cell lymphoma 10 (Bcl10) is a scaffolding protein that functions as an upstream regulator of NF-κB signaling by forming a complex with Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and CARD-coiled coil protein family. This study showed that Bcl10 was involved in type I interferon (IFN) expression in response to DNA virus infection and that Bcl10-deficient mice were more susceptible to Herpes simplex virus 1 (HSV-1) infection than control mice. Mechanistically, DNA virus infection can trigger Bcl10 recruitment to the STING-TBK1 complex, leading to Bcl10 phosphorylation by TBK1. The phosphorylated Bcl10 undergoes droplet-like condensation and forms oligomers, which induce TBK1 phosphorylation and translocation to the perinuclear region. The activated TBK1 phosphorylates IRF3, which induces the expression of type I IFNs. This study elucidates that Bcl10 induces an innate immune response by undergoing droplet-like condensation and participating in signalosome formation downstream of the cGAS-STING pathway.

Nicotinamide N-Methyltransferase Remodeled Cell Metabolism and Aggravated Proinflammatory Responses by Activating STAT3/IL1ß/PGE2 Pathway.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic methyltransferase, catalyzing N-methylation of nicotinamide (NAM) to form 1-methylnicotinamide (1-MNAM), in which S-adenosyl-l-methionine (SAM) is the methyl donor. It has been well documented that NNMT is elevated in multiple cancers and promotes tumor aggressiveness. In the present study, we investigated the effects of NNMT overexpression on cellular metabolism and proinflammatory responses. We found that NNMT overexpression reduced NAD+ and SAM levels, and activated the STAT3 signaling pathway. Consequently, STAT3 activation upregulated interleukin 1ß (IL1ß) and cyclooxygenase-2 (COX2), leading to prostaglandin E2 (PGE2) accumulation. On the other hand, NNMT downregulated 15-hydroxyprostaglandin dehydrogenase (15-PGDH) which catalyzes PGE2 into inactive molecules. Moreover, secretomic data indicated that NNMT promoted secretion of collagens, pro-inflammatory cytokines, and extracellular matrix proteins, confirming NNMT aggravated inflammatory responses to promote cell growth, migration, epithelial-mesenchymal transition (EMT), and chemoresistance. Taken together, we showed that NNMT played a pro-inflammatory role in cancer cells by activating the STAT3/IL1ß/PGE2 axis and proposed that NNMT was a potential therapeutic target for cancer treatment.

Lentiviral vector-based xenograft tumors as candidate reference materials for detection of HER2-low breast cancer.

The human epidermal growth factor receptor 2 (HER2) is an important biomarker that plays a pivotal role in therapeutic decision-making for patients with breast cancer (BC). Patients with HER2-low BC can benefit from new HER2 targeted therapy. For ensuring the accurate and reproducible detection of HER2-low cancer, reliable reference materials are required for monitoring the sensitivity and specificity of detection assays. Herein, a lentiviral vector was used to transduce the HER2 gene into MDA-MB-231 cells that exhibited low HER2 density, and the cells were characterized by droplet digital PCR to accurately determine the copy number variation. Then, the formalin-fixed paraffin-embedded (FFPE) samples from xenografts were prepared and evaluated for suitability as candidate reference materials by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The FFPE reference materials were selected on the basis of IHC score of 2+ and negative FISH result to meet the requirement for HER2-low BC detection. Furthermore, the FFPE reference materials exhibited typical histological structures that resembled the clinical BC specimens. These novel FFPE reference materials displayed the high stability and homogeneity, and they were produced in high quantity. In summary, we generated high-quality reference materials for internal quality control and proficiency testing in HER2-low detection.

Forming cytoplasmic stress granules PURα suppresses mRNA translation initiation of IGFBP3 to promote esophageal squamous cell carcinoma progression.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment.

Albumin binding revitalizes NQO1 bioactivatable drugs as novel therapeutics for pancreatic cancer.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) tumors compared to the associated normal tissues. NQO1 bioactivatable drugs, such as ß-lapachone (ß-lap), can be catalyzed to generate reactive oxygen species (ROS) for direct tumor killing. However, the extremely narrow therapeutic window caused by methemoglobinemia and hemolytic anemia severely restricts its further clinical translation despite considerable efforts in the past 20 years. Previously, we demonstrated that albumin could be utilized to deliver cytotoxic drugs selectively into KRAS-mutant PDAC with a much expanded therapeutic window due to KRAS-enhanced macropinocytosis and reduced neonatal Fc receptor (FcRn) expression in PDAC. Herein, we analyzed the expression patterns of albumin and FcRn across major organs in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice. The tumors were the predominant tissues with both elevated albumin and reduced FcRn expression, thus making them an ideal target for albumin-based drug delivery. Quantitative proteomics analysis of tissue samples from 5 human PDAC patients further confirmed the elevated albumin/FcRn ratio. Given such a compelling biological rationale, we designed a nanoparticle albumin-bound prodrug of ß-lap, nab-(pro-ß-lap), to achieve PDAC targeted delivery and expand the therapeutic window of ß-lap. We found that nab-(pro-ß-lap) uptake was profoundly enhanced by KRAS mutation. Compared to the solution formulation of the parent drug ß-lap, nab-(pro-ß-lap) showed enhanced safety due to much lower rates of methemoglobinemia and hemolytic anemia, which was confirmed both in vitro and in vivo. Furthermore, nab-(pro-ß-lap) significantly inhibited tumor growth in subcutaneously implanted KPC xenografts and enhanced the pharmacodynamic endpoints (e.g., PARP1 hyperactivation, γ-H2AX). Thus, nab-(pro-ß-lap), with improved safety and antitumor efficacy, offers a drug delivery strategy with translational viability for ß-lap in pancreatic cancer therapy.

Flavonoid 4,4'-dimethoxychalcone induced ferroptosis in cancer cells by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH.

Flavonoids are widely distributed in plants as secondary metabolites and have various biological benefits such as anti-tumor, anti-oxidant, anti-inflammatory and anti-aging. We previously reported that 4,4'-dimethoxychalcone (DMC) suppressed cancer cell proliferation by aggravating oxidative stress and inducing G2/M cell cycle arrest. In the present study, we explored the underlying mechanisms by which DMC inhibited cancer cell growth. Given that ferrochelatase (FECH) is a potential target of DMC identified by thermal proteome profiling (TPP) method, herein, we confirmed that DMC inhibited the enzymatic activity of FECH. Furthermore, we proved that DMC induced Keap1 degradation via ubiquitin-proteasome system, which led to the nuclear translocation of Nrf2 and upregulated Nrf2 targeted gene HMOX1. FECH inhibition and HMOX1 upregulation resulted in iron overload and triggered ferroptosis in cancer cells. Collectively, we revealed that DMC induced ferroptosis by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH. Our findings indicate that FECH contributes to the non-canonical ferroptosis induction, shed light on the mechanisms of DMC inhibiting cancer cell growth, and set an example for studying biological functions of flavonoids.

RIOK1 mediates p53 degradation and radioresistance in colorectal cancer through phosphorylation of G3BP2.

RIO Kinase 1 (RIOK1) is involved in various pathologies, including cancer. However, the role of RIOK1 in radioresistance of colorectal cancer (CRC) remains largely unknown. In this study, we reported that RIOK1 was overexpressed in rectal cancer tissue with weaker tumor regression after neoadjuvant chemoradiotherapy (neoCRT). Moreover, higher RIOK1 expression predicted a poor prognosis in patients with rectal cancer. Blockade of RIOK1 using Toyocamycin, a pharmacological inhibitor of RIOK1, or by knocking down its expression, decreased the resistance of CRC cells to radiotherapy in vitro and in vivo. A mechanistic study revealed that RIOK1 regulates radioresistance by suppressing the p53 signaling pathway. Furthermore, we found that RIOK1 and Ras-GAP SH3 domain binding protein 2 (G3BP2) interact with each other. RIOK1 phosphorylates G3BP2 at Thr226, which increases the activity of G3BP2. RIOK1-mediated phosphorylation of G3BP2 facilitated ubiquitination of p53 by murine double minute 2 protein (MDM2). Altogether, our study revealed the clinical significance of RIOK1 in CRC, and therapies targeting RIOK1 might alleviate the CRC tumor burden in patients.

Nicotinamide reprograms adipose cellular metabolism and increases mitochondrial biogenesis to ameliorate obesity.

Obesity poses a global health challenge and is a major risk factor for diabetes mellitus, cardiovascular diseases, hypertension, stroke and certain kinds of cancers. Although the effects of nicotinamide (NAM) on liver metabolism and diseases were well documented, its effects on adipose tissue are yet to be characterized. Herein, we found that NAM supplementation significantly reduced fat mass and improved glucose tolerance in obese mice. Proteomic analysis revealed that NAM supplementation upregulates mitochondrial proteins while quantitative polymerase chain reaction showed that PPARα and PGC1α were both upregulated in adipose tissues, proposing that NAM increased mitochondrial biogenesis in adipose tissue. Indeed, NAM treatment increased proteins related to mitochondrial functions including oxidative phosphorylation, fatty acid oxidation, and TCA cycle. Furthermore, isotope-tracing assisted metabolic profiling revealed that NAM activated NAMPT and increased cellular NAD+ level by 30%. Unexpectedly, we found that NAM also increased glucose derived amino acids to enhance glutathione synthesis for maintaining cellular redox homeostasis. Taken together, our results demonstrated that NAM reprogramed cellular metabolism, enhanced adipose mitochondrial functions to ameliorate symptoms associated with obesity.

Ablation of Proton/Glucose Exporter SLC45A2 Enhances Melanosomal Glycolysis to Inhibit Melanin Biosynthesis and Promote Melanoma Metastasis.

Sequence variation in SLC45A2 are responsible for oculocutaneous albinism type 4 in many species and are associated with melanoma susceptibility, but the molecular mechanism is unclear. In this study, we used Slc45a2-deficient melanocyte and mouse models to elucidate the roles of SLC45A2 in melanogenesis and melanoma metastasis. We found that the acidified cellular environment impairs the activity of key melanogenic enzyme tyrosinase in Slc45a2-deficient melanocytes. SLC45A2 is identified as a proton/glucose exporter in melanosomes, and its ablation increases the acidification of melanosomal pH through enhanced glycolysis. Intriguingly, 13C-glucose-labeled metabolic flux and biochemical assays show that melanosomes are active glucose-metabolizing organelles, indicating that elevated glycolysis mainly occurs in melanosomes owing to Slc45a2 deficiency. Moreover, Slc45a2 deficiency significantly upregulates the activities of glycolytic enzymes and phosphatidylinositol 3-kinase/protein kinase B signaling to promote glycolysis-dependent survival and metastasis of melanoma cells. Collectively, our study reveals that the proton/glucose exporter SLC45A2 mediates melanin synthesis and melanoma metastasis primarily by modulating melanosomal glucose metabolism.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates.

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

Profiling Glutathionylome in CD38-Mediated Epithelial-Mesenchymal Transition.

Protein S-glutathionylation is an important posttranslational modification that regulates various cellular processes. However, changes in glutathionylome in epithelial-mesenchymal transition (EMT), a crucial cellular process for embryonic development, wound healing, and carcinoma progression and metastasis, have not been fully characterized. Our previous study revealed that CD38 overexpression decreased cellular nicotinamide adenine dinucleotide (NAD+) levels and caused cells to undergo EMT. In the present study, we engineered a cell system in which the glutathione synthetase (GS) mutant was expressed that catalyzed the formation of a glutathione analogue from azido-alanine to profile changes of glutathionylome in CD38-overexpressing cells. We identified 1298 glutathionylated proteins and revealed that proteins with changed glutathionylation levels involved in EMT associated pathways including epithelial adherens junction, actin cytoskeleton, and integrin signaling. Moreover, the glutathionylation level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was increased in CD38-overexpressing cells. We further demonstrated that glutathionylation of Cys63 residue in 15-PGDH led to decreased enzymatic activity that could promote EMT by increasing prostaglandin E2 (PGE2). Taken together, these results indicate that the clickable glutathione is an effective probe for glutathionylome profiling, and glutathionylation of 15-PGDH on Cys63 inhibits its enzymatic activity to promote EMT.