Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the ß-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

Relationship between Chemotherapy-Induced Diarrhea and Intestinal Microbiome Composition.

BACKGROUND AND AIM: Fluoropyrimidines (FPs) are key drugs in many chemotherapy regimens; however, recipients are often prone to diarrhea due to gastrointestinal toxicity. Disruption of the intestinal epithelial barrier function by FPs leads to dysbiosis, which may exacerbate intestinal epithelial cell damage as a secondary effect and trigger diarrhea. However, despite studies on chemotherapy-induced changes in the intestinal microbiome of humans, the relationship between dysbiosis and diarrhea is unclear. In this study, we aimed to investigate the relationship between chemotherapy-induced diarrhea and the intestinal microbiome. METHODS: We conducted a single-center prospective observational study. Twenty-three patients who received chemotherapy, including FPs as first-line chemotherapy for colorectal cancer, were included. Stool samples were collected before the start of chemotherapy and after one cycle of treatment to analyze intestinal microbiome composition and perform PICRUSt predictive metagenomic analysis. RESULTS: Gastrointestinal toxicity was observed in 7 of 23 patients (30.4%), diarrhea was observed in 4 (17.4%), and nausea and anorexia were observed in 3 (13.0%). In 19 patients treated with oral FPs, the α diversity of the microbial community decreased significantly following chemotherapy only in the diarrheal group. At the phylum level, the diarrheal group showed a significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Bacteroidetes with chemotherapy (p = 0.013 and 0.011, respectively). In the same groups, at the genus level, Bifidobacterium abundance was significantly decreased (p = 0.019). In contrast, in the non-diarrheal group, Actinobacteria abundance increased significantly with chemotherapy at the phylum level (p = 0.011). Further, Bifidobacterium, Fusicatenibacter, and Dorea abundance significantly increased at the genus level (p = 0.006, 0.019, and 0.011, respectively). The PICRUSt predictive metagenomic analysis revealed that chemotherapy caused significant differences in membrane transport in KEGG pathway level 2 and in 8 KEGG pathway level 3, including transporters and oxidative phosphorylation in the diarrhea group. CONCLUSION: Organic-acid-producing bacteria seem to be involved in diarrhea associated with chemotherapy, including FPs.

GEF mechanism revealed by the structure of SmgGDS-558 and farnesylated RhoA complex and its implication for a chaperone mechanism.

SmgGDS has dual functions in cells and regulates small GTPases as both a guanine nucleotide exchange factor (GEF) for the Rho family and a molecular chaperone for small GTPases possessing a C-terminal polybasic region followed by four C-terminal residues called the CaaX motif, which is posttranslationally prenylated at its cysteine residue. Our recent structural work revealed that SmgGDS folds into tandem copies of armadillo-repeat motifs (ARMs) that are not present in other GEFs. However, the precise mechanism of GEF activity and recognition mechanism for the prenylated CaaX motif remain unknown because SmgGDS does not have a typical GEF catalytic domain and lacks a pocket to accommodate a prenyl group. Here, we aimed to determine the crystal structure of the SmgGDS/farnesylated RhoA complex. We found that SmgGDS induces a significant conformational change in the switch I and II regions that opens up the nucleotide-binding site, with the prenyl group fitting into the cryptic pocket in the N-terminal ARMs. Taken together, our findings could advance the understanding of the role of SmgGDS and enable drug design strategies for targeting SmgGDS and small GTPases.

Structure-based analysis of the guanine nucleotide exchange factor SmgGDS reveals armadillo-repeat motifs and key regions for activity and GTPase binding.

Small GTPases are molecular switches that have critical biological roles and are controlled by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs). The smg GDP dissociation stimulator (SmgGDS) protein functions as a GEF for the RhoA and RhoC small GTPases. SmgGDS has various regulatory roles, including small GTPase trafficking and localization and as a molecular chaperone, and interacts with many small GTPases possessing polybasic regions. Two SmgGDS splice variants, SmgGDS-558 and SmgGDS-607, differ in GEF activity and binding affinity for RhoA depending on the lipidation state, but the reasons for these differences are unclear. Here we determined the crystal structure of SmgGDS-558, revealing a fold containing tandem copies of armadillo repeats not present in other GEFs. We also observed that SmgGDS harbors distinct positively and negatively charged regions, both of which play critical roles in binding to RhoA and GEF activity. This is the first report demonstrating a relationship between the molecular function and atomic structure of SmgGDS. Our findings indicate that the two SmgGDS isoforms differ in GTPase binding and GEF activity, depending on the lipidation state, thus providing useful information about the cellular functions of SmgGDS in cells.

Comparing the Effects of Anti-TNF Agent and Ustekinumab on Small Bowel Inflammation in Crohn's Disease: Inverse Probability Weighting With Stabilized Weights of Propensity Scores.

Background: Endoscopic mucosal healing serves as a critical predictor for achieving long-term remission in Crohn's disease treatment. Recent data indicate that the effectiveness of healing varies based on the location of gastrointestinal inflammation. Additionally, reports suggest that antitumor necrosis factor-α (anti-TNF-α) agents exhibit reduced efficacy in treating small intestinal inflammation compared to colorectal inflammation. Conversely, limited research exists regarding the impact of the anti-IL12/23 agent ustekinumab (UST) on small intestinal inflammation. This study aimed to compare the effects of anti-TNF-α agents and UST on small intestinal inflammation using propensity score analysis. Methods: This retrospective observational study involved 70 patients with Crohn's disease who had inflammation in the small intestine and had initiated treatment with either anti-TNF agents or UST between March 2015 and August 2021. Endoscopic findings were evaluated before treatment commencement and at 1-2 years post-treatment initiation. The propensity score was employed to compare the efficacy of TNF agents and UST on small bowel inflammation. Results: Ustekinumab exhibited greater improvement in the small intestinal endoscopy score than anti-TNF-α antibodies according to the propensity score analysis (inverse probability weighting; P = .0448). However, no significant disparity was observed in the overall improvement of endoscopic scores between UST and anti-TNF-α antibodies (P = .5938). Conclusions: This study suggests that UST might be more effective than anti-TNF-α agents in treating small intestinal inflammation in Crohn's disease.

Gut microbiota shifts from onset to remission in immune checkpoint inhibitor-induced enterocolitis: a case report.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are crucial in cancer treatment; however, they carry the risk of immune-related adverse events (irAEs), such as enteritis. CASE PRESENTATION: This study investigated the role of the gut microbiota during the onset and remission of irAE enteritis in a patient with stage IV melanoma undergoing anti-PD-1 and anti-CTLA-4 therapy. Following commencement of ICI treatment, the patient developed severe diarrhea and was diagnosed with grade 3 irAE enteritis. Steroid and probiotic treatments provided swift symptom relief and remission, as confirmed by reduced fecal calprotectin levels and gastrointestinal imaging. Microbiota diversity analysis conducted via 16S rRNA gene sequencing identified a decrease in Streptococcus prevalence with improvement in enteritis symptoms. Conversely, genera Fusobacterium, Faecalibacterium, Bacteroides, Prevotella, and Bifidobacterium showed increased representation after remission. These genera are associated with anti-inflammatory properties and fibrous substrate degradation, aiding gut health. Immunological assessment demonstrated fluctuations in cytokine expression and the modulation of costimulatory molecules, aligning with therapeutic interventions and microbiota alterations. CONCLUSIONS: Our findings indicate a significant correlation between gut microbiota and immune responses in irAE enteritis. This underscores the potential utility of microbiome profiling in predicting irAE occurrence and in providing treatment strategies, thereby promoting a more comprehensive approach to managing the adverse effects of ICIs.

Serum IL-13 Predicts Response to Golimumab in Bio-Naïve Ulcerative Colitis.

A certain number of patients with ulcerative colitis (UC) are refractory to anti-TNF-α antibodies; biomarkers are thus needed to predict treatment efficacy. This study aimed to evaluate whether serum biomarkers that were reported to be associated with UC or anti-TNF-α antibody could predict the response to golimumab, a human anti-TNF-α monoclonal antibody, in bio-naïve patients with UC. We prospectively enrolled 23 consecutive patients with UC who were treated with golimumab. Serum samples were collected before the first golimumab dose. Eleven molecules were measured by electrochemiluminescence (ECL) or enzyme-linked immunosorbent assay (ELISA) and their association with efficacy after 10 weeks of golimumab treatment. Among the serum biomarkers, IL-13 levels were significantly higher in the non-remission group than in the remission group (p = 0.014). IL-15 levels were significantly lower in the non-response group than in the response group (p = 0.04). For clinical remission at week 10, the IL-13 0.20 concentration of pg/mL was associated with a sensitivity and specificity of 82.4% and 83.3%, respectively. Serum IL-13 may be a biomarker to predict golimumab efficacy in biologic-naïve patients with UC, and thus may help to tailor personalized treatment strategies.

Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H2O2-dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H2O2/Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases.