Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) represents a family of important transcription factors in innate immunity. We have previously reported that the gastric pathogen Helicobacter pylori needs the actin-binding protein cortactin for efficient interleukin-8 (IL-8) secretion, which requires NF-κB activation. However, it remained unknown, which exact cortactin signaling mechanism contributes to IL-8 release. In fact, H. pylori profoundly activates NF-κB in wild-type AGS gastric epithelial cells by the effector molecule adenosine diphosphate (ADP)-β-d-manno-heptose (ADPH) in a type IV secretion system-dependent manner. However, the injected CagA protein might contribute to NF-κB activation. The ADPH-stimulated canonical NF-κB cascade involves alpha-kinase 1 and adapter protein TRAF-interacting protein with forkhead-associated domain (TIFA) to activate inhibitor of kappa B (IκB) kinases (IKKs), followed by phosphorylation-dependent degradation of IκBα and subsequent nuclear translocation of p65 NF-κB and IL-8 release. Here, we show that infection of cortactin knockout cells leads to reduced activation of focal adhesion kinase (FAK) and c-Sarcoma (Src) kinase resulting in diminished phosphorylation of IKKβ at tyrosine residue 199 and subsequently phosphorylation of p65 at serine residue 536, both of which are associated with downregulated NF-κB activity. Our results were further supported using FAK and TIFA knockout cells and treatments with purified ADPH and overexpression of CagA, showing cumulative effects in wild-type, but not in knockout cells. These data demonstrate that ADPH-dependent NF-κB activation and IL-8 secretion are enhanced by CagA. Together, we present here a novel CagA>cortactin>FAK>Src>IKKβ signaling cascade, contributing to proinflammatory responses by H. pylori.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, underscoring the urgent need for precise and personalized therapeutic strategies. Globo-H has emerged as a clinically relevant glycan target with promising diagnostic and therapeutic utility across multiple cancer types. In this study, we stratified colorectal cancer patients into Globo-H-high and Globo-H-low groups using a histology-based classification, followed by RNA-sequencing analyses to elucidate the key signaling pathways associated with Globo-H overactivation. Among the 31 genes that were identified to meet the Globo-H histology criterion, DUSP14 (dual specificity phosphatase 14) emerged as a promising pharmacological target associated with Globo-H abundance. DUSP14 is an underexplored but pharmacologically actionable therapeutic target. DUSP14 protein in colon cancer cells is inversely correlated with total Transforming growth factor-β-activated kinase 1 (TAK1) protein. The druggability of DUSP14 was demonstrated through in vitro using cell lines and patient-derived organoids (PDO). These results enhance current diagnostic frameworks and provide a foundation for developing novel targeted therapies. Further, in vivo studies are warranted to evaluate the potential of Globo-H targeting in combination with standard treatment regimens. Overall, our work highlights the value of integrating PDO-based functional assays with molecular profiling to uncover and validate actionable targets for CRC theranostics.

Abstract In the gastrointestinal tract, Wnt and BMP signals control Lgr5 ⁺ stem cell activity during homeostasis, whereas injury elicits an Lgr5 -independent, fetal-like regenerative program driven by YAP. Helicobacter pylori ( H. pylori ) infection activates YAP, but whether fetal-like reprogramming contributes to gastric pathology, and what drives it, has remained unclear. Here we show that H. pylori -induced gland hyperplasia is accompanied by YAP-dependent fetal-like transcriptional response and loss of epithelial BMP signaling. Epithelial BMP inhibition alone is sufficient to induce this program in vivo, through an epithelial–immune–stromal cascade: BMP-deficient epithelial cells secrete chemokines that recruit IL-1β-producing immune cells, and IL-1β drives enrichment of pro-regenerative fibroblasts producing prostaglandin E2. In gastric epithelial–stromal assembloids, IL-1β elicits stromal prostaglandin E2 production and subsequent epithelial YAP activation. Stromal deletion of the IL-1 receptor abrogates H. pylori -driven reprogramming and pathology. These data define a cascade that converts BMP loss into a fetal-like regenerative state and shapes H. pylori -associated gastric disease.

Caspase 3 is a key executioner of apoptotic cell death and contributes to intestinal epithelial homeodynamics. Apoptotic dysregulation has been implicated in Crohn’s disease, yet data on caspase 3 expression across disease activity states remain limited. This study analyzed caspase 3 expression in intestinal biopsies from Crohn’s disease patients. Paraffin-embedded biopsies from 289 individuals were examined, including active disease (mild and severe inflammation), upper gastrointestinal involvement, remission and non-inflamed tissue. Expression in epithelial and immune cells was assessed by immunohistochemistry and scored using the Remmele immunoreactive score (IRS). Caspase 3 expression levels in epithelial cells increased in cases of severe inflammation (p=0.012), and immune cells exhibited even more pronounced expression levels (p<0.001). These findings suggest that caspase 3 expression in epithelial and immune cells may help to distinguish between mild and severe inflammation.

BACKGROUND: Ubiquitin-specific protease 19 (USP19) is a deubiquitinylase (DUB) that is part of the USP family, the largest group of DUBs in humans. Growing evidence has indicated that USP19 is involved in tumor progression and serves as a new prognostic marker for various malignant disorders. Interestingly, USP19 has been shown to have both promoting and inhibiting effects on the onset and development of different neoplasms, depending on the specific tissue type. DUBs including USP19 affect a variety of cell functions including apoptotic cell death. Herein, caspase 7 acts as a key executioner in apoptosis, and its expression levels serve as a prognostic and diagnostic marker in various cancers. This study analysed the expression of USP19 and caspase 7 in gastric pathology along the progression of stomach cells to gastric adenocarcinoma (Correa cascade). METHODS: We analysed the expression and subcellular localization of USP19 and caspase 7 by immunohistochemistry (IHC) in 296 paraffin-embedded human gastric tissue samples. The cohort included various gastric conditions such as autoimmune gastritis (A-gastritis), Helicobacter pylori gastritis (HP-gastritis), chemical gastropathy (C-gastritis), adenoma, and adenocarcinoma, using gastric mucosa without pathological changes as a reference point. RESULTS: We observed a significant upregulation of USP19 expression in HP-gastritis, adenoma and adenocarcinoma. In contrast, caspase 7 was significantly upregulated in A-gastritis and HP-gastritis and significantly downregulated in both adenoma and adenocarcinoma. CONCLUSIONS: USP19 and caspase 7 showed distinct expression patterns across different gastric pathologies. Both USP19 and caspase 7 overexpression maybe associated with inflammation, while USP19 overexpression and no caspase 7 expression could indicate neoplastic transformation. This inverse expression may help distinguish early and late neoplastic epithelial changes in chronic gastritis.

Deubiquitinases (DUBs) regulate substrate ubiquitination, thereby modulating signal transduction, trafficking, and proteasomal degradation. They have emerged as promising therapeutic targets owing to their involvement in a variety of pathological conditions. However, the limited availability of DUB inhibitors with adequate specificity, safety, and efficacy remains a major barrier to successful clinical translation. Recent studies indicate that exploring both allosteric and covalent inhibition strategies together with integrated computational workflows could address this gap. In this review, we summarize current developments concerning the chemistry of small-molecule DUB inhibitors, improved computational discovery workflows, and the preclinical evaluation of candidate compounds.

Ubiquitinylation of proteins regulates manifold processes and is reversed by deubiquitinylating enzymes (DUBs), which are therefore implicated in a plethora of cellular processes. DUBs are frequently upregulated in many diseases, while in a few cases downregulation of DUBs is associated with disease progression. This review focuses on the involvement of DUBs in the development and progression of gastrointestinal diseases with a particular emphasis on hepatic steatosis and hepatocellular, cholangio-, esophageal, gastric, colorectal, and pancreatic ductal carcinomas. In addition, pathogens that trigger the activity of several DUBs and thus suppress the immune response and cell survival are discussed. Finally, we highlight recent approaches made towards the therapeutic treatment of gastrointestinal diseases using DUB inhibitors.

A20, an ubiquitin-editing enzyme, plays a pivotal role in regulating cell signaling and immune responses. Dysregulated A20 expression has been associated with various pathological conditions, including inflammatory diseases and malignancies, where its expression levels often correlate with differing prognoses in solid tumors. This study aimed to explore the expression and cellular localization of A20 in both nonpathological and diseased human gastric tissues to gain deeper insights into its involvement in gastric pathologies. We analyzed paraffin-embedded gastric tissue samples from 326 patients. A20 expression was assessed using immunohistochemistry (IHC) with results categorized according to the Remmele and Stegner immunoreactive score (IRS). The study compared A20 expression across a spectrum of gastric pathologies, including Helicobacter pylori (HP) gastritis, autoimmune gastritis (A-gastritis), reactive gastropathy (C-gastritis), Ex-HP-gastritis, adenomas, and adenocarcinomas, with nonpathological gastric mucosa serving as a baseline. Our findings demonstrate a significant increase in A20 expression in HP-gastritis (p = 0.019), A-gastritis (p = 0.001), adenomas (p < 0.001), and adenocarcinomas (p < 0.001). Conversely, no significant differences in A20 expression were observed in C-gastritis or Ex-HP-gastritis cases.

Despite a decline in global incidence, gastric cancer (GC) remains a major health concern. The development of GC is a sequential, multistage maladaptive process involving numerous different factors. Understanding the complexity of GC development is crucial for early detection, effective treatment, and, ultimately, prevention. In this respect, identifying the impact of risk factors contributing to the emergence or progression of GC, such as Helicobacter pylori infection, host and bacterial genetics, alcohol consumption, smoking, and preserved foods, will aid in combatting this disease. In this review, we focus on recent developments in understanding the role of the microbiome, dysfunctional molecular pathways, and immune evasion in gastric pathophysiology. We also highlight challenges and advances in treatment of GC.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

In atopic dermatitis (AD), lesional skin is frequently colonized by Staphylococcus aureus, which promotes clinical symptoms of the disease. The inflammatory milieu in the skin is characterized by a Th2 response, including M2 macrophages, which cannot eradicate S. aureus. Therefore, repolarization of macrophages toward the M1 phenotype may foster control of S. aureus. Our data show that the deubiquitinating enzyme cylindromatosis (CYLD) is strongly expressed in macrophages of AD patients and prevents the clearance of S. aureus. Mechanistically, CYLD impaired M1 macrophage polarization by K63-specific deubiquitination of STAT1 and activation of the NF-κB pathway via its interaction with TRAF6, NEMO, and RIPK2. Inhibition of STAT1 and NF-κB, independently, abolished the differences between S. aureus-infected CYLD-deficient and CYLD-competent M1 macrophages. Infection of Cyld-deficient and wild-type mice with S. aureus confirmed the protective CYLD function. Collectively, our study shows that CYLD impairs the control of S. aureus in macrophages of AD patients, identifying CYLD as a potential therapeutic target.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that has colonized nearly 30% of the human population and can cause life-threatening infections. S. aureus exports a variety of virulence factors, such as a novel set of extracellular serine protease-like proteins (Spls). Spls are expressed by most clinical isolates of S. aureus, but their pathophysiological substrates and role during the infection are largely unknown. Here we characterized the substrate and cleavage specificity of recombinantly expressed SplA and SplB proteins. We identified a group of ubiquitin or ubiquitin-like modifying enzymes including deubiquitinating enzymes from human as well as from bacterial sources to be so far unknown SplA and SplB substrates. Distinct cleavage sites within these substrates for SplA (YLY↓T, FMY↓N) and SplB (VCD↓S) were identified by mass spectrometry and confirmed by site-directed mutagenesis of the target proteins. Since many cellular immune signaling pathways are tightly regulated by ubiquitination, the specific cleavage of ubiquitin modifying enzymes strongly suggests a specific role of Spls in manipulating immune signaling and in competing with other bacteria.

Recent discoveries revealed mechanistic insights into the control of adipogenesis by the Constitutive Photomorphogenesis 9 Signalosome (CSN) and its variants, CSNCSN7A and CSNCSN7B, which differ in the paralog subunits, CSN7A and CSN7B. CSNCSN7A and CSNCSN7B variants form permanent complexes with cullin-RING-ubiquitin ligases 3 and 4A (CRL3 and CRL4A), respectively. These complexes can be found in most eukaryotic cells and represent a critical reservoir for cellular functions. In an early stage of adipogenesis, mitotic clonal expansion (MCE), CSN-CRL1, and CSNCSN7B-CRL4A are blocked to ubiquitinate the cell cycle inhibitor p27KIP, leading to cell cycle arrest. In addition, in MCE CSN-CRL complexes rearrange the cytoskeleton for adipogenic differentiation and CRL3KEAP1 ubiquitylates the inhibitor of adipogenesis C/EBP homologous protein (CHOP) for degradation by the 26S proteasome, an adipogenesis-specific proteolysis. During terminal adipocyte differentiation, the CSNCSN7A-CRL3 complex is recruited to a lipid droplet (LD) membrane by RAB18. Currently, the configuration of the substrate receptors of CSNCSN7A-CRL3 on LDs is unclear. CSNCSN7A-CRL3 is activated by neddylation on the LD membrane, an essential adipogenic step. Damage to CSN/CUL3/CUL4A genes is associated with diverse diseases, including obesity. Due to the tremendous impact of CSN-CRLs on adipogenesis, we need strategies for adequate treatment in the event of malfunctions.

Cezanne-2 (Cez2) is a deubiquitinylating (DUB) enzyme involved in the regulation of ubiquitin-driven cellular signaling and selectively targets Lys11-linked polyubiquitin chains. As a representative member of the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic isopeptide bond cleavage; however, its exact catalytic mechanism is not yet resolved. In this work, we used different computational approaches to get molecular insights into the Cezanne-2 catalytic mechanism. Extensive molecular dynamics (MD) simulations were performed for 12 mu s to model free Cez2 and the diubiquitin (diUb) substrate-bound protein-protein complex in two different charge states of Cez2, each corresponding to a distinct reactive state in its catalytic cycle. The simulations were analyzed in terms of the relevant structural parameters for productive enzymatic catalysis. Reactive diUb-Cez2 complex configurations were identified, which lead to isopeptide bond cleavage and stabilization of the tetrahedral oxyanion intermediate. The reliability of these complexes was further assessed by quantum mechanics/molecular mechanics (QM/MM) optimizations. The results show that Cez2 follows a modified cysteine protease mechanism involving a catalytic Cys210/His367 dyad, with the oxyanion hole to be a part of the "C-loop," and polarization of His367 by the formation of a strictly conserved water bridge with Glu173. The third residue has a dual role in catalysis as it mediates substrate binding and polarization of the catalytic dyad. A similar mechanism was identified for Cezanne-1, the paralogue of Cez2. In general, our simulations provide valuable molecular information that may help in the rational design of selective inhibitors of Cez2 and closely related enzymes.

In 1983, Linus Pauling and colleagues reported about enhanced antitumor activity of the Cu(II) complex of the simplest ATCUN (amino terminal Cu(II) and Ni(II)-binding motif) peptide (NH2-Gly-Gly-His-COOH, GGH) in the presence of ascorbate as an additive. In the following 4 decades, structural modifications of this complex were implemented, however, anticancer activity could not be significantly increased. This has led to neglecting the ATCUN motif and its Cu(II) complexes as potential chemotherapeutic agents. Furthermore, the addition of ascorbate with its positive effect on the anticancer activity has fallen into oblivion. In this work, we compared Cu(II) GGH with Cu(II) ATCUN peptides bearing β-Ala instead of Gly at the 2nd position of the peptide sequence regarding their in vitro complex stability and cytotoxicity (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and annexin V-FITC (fluorescein isothiocyanate) apoptosis assay) towards three cancer cell lines (AGS, HeLa and NCI-N87). Such an exchange of amino acids led to an up to three-fold higher cytotoxic effect in the presence of ascorbate. We thus achieved a significant increase in the otherwise moderate cytotoxicity of Cu(II) ATCUN-like complexes. Lipophilicity assays (n-octanol/water coefficient, log P values) of the studied complexes were used to evaluate differences in the antiproliferative activity.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.