Regulatory Mechanisms Research Articles

Dopamine Regulates Its Own Synthesis via MdORG2 to Improve Low-Nitrogen Tolerance in Apple Plants.

Nitrogen (N) is crucial for plant growth and development. Exogenous dopamine has been shown to improve the N-deficiency tolerance of apple. However, the potential regulatory mechanisms by which dopamine mitigates low-N stress remain unclear. Our data indicated that the dopamine levels in apple (Malus domestica) were elevated by the overexpression (OE) of MdTYDC, which encodes tyrosine decarboxylase, a key enzyme in dopamine biosynthesis. The photosynthetic capacity of the OE lines was enhanced, and the root system was more extensive under low-N stress compared with the wild-type (WT) plants. This enhancement contributed to a greater net nitrate influx at the root surface in the OE lines compared with the WT. Transcriptomic and carbohydrate analyses suggested that the OE of MdTYDC in apple enhanced N-deficiency tolerance by promoting the expression of carbohydrate-related genes, which increased the content of soluble sugars and sorbitol. Both exogenous dopamine and MdTYDC OE activated the expression of MdORG2 (a bHLH transcription factor), which, in turn, directly binds to the promoter of MdTYDC, activating its expression, increasing dopamine levels, and consequently conferring strong low-N tolerance in apple. Thus, this reveals the molecular pathways by which dopamine regulates low-N tolerance in apple through pathways involving MdTYDC and MdORG2.

Stabilization of hypoxia-inducible factor 1α and regulation of specific gut microbes by EGCG contribute to the alleviation of ileal barrier disorder and obesity.

Tea polyphenols have a regulatory effect on metabolic-related diseases, however, the underlying mechanism remains elusive. Our study aims to explore the dietary intervention effect of Epigallocatechin gallate (EGCG), the major polyphenol in green tea, on obesity and intestinal barrier disorders in mice fed a high-fat diet. By supplementing with 50 mg kg-1 EGCG, we observed a significant amelioration in body weight gain, fat accumulation, and liver dysfunction. Furthermore, EGCG modulated the HFD-induced metabolomic alterations. In particular, EGCG intervention restored the ileal barrier by enhancing the expression of tight junction proteins and antimicrobial peptides. At the mechanistic level, EGCG treatment stabilized hypoxia-inducible factor 1α (HIF1α) both in vitro and in vivo. Meanwhile, EGCG significantly increased the abundance of Dubosiella and Akkermansia, along with the elevated SCFA contents. These findings suggest that the ability of EGCG to stabilize HIF1α and regulate specific gut microbes is pivotal in mitigating ileal barrier dysfunction and obesity. Moreover, serum metabolomics revealed potential biomarkers following EGCG intervention. This study supports the intake of EGCG or green tea in obesity management and offers a novel perspective for investigating the metabolic regulatory mechanism of other dietary polyphenols.

Ubiquitylomics: An Emerging Approach for Profiling Protein Ubiquitylation in Skeletal Muscle.

Skeletal muscle is a highly adaptable tissue, finely tuned by various physiological and pathological factors. Whilst the pivotal role of skeletal muscle in overall health is widely acknowledged, unravelling the underlying molecular mechanisms poses ongoing challenges. Protein ubiquitylation, a crucial post-translational modification, is involved in regulating most biological processes. This widespread impact is achieved through a diverse set of enzymes capable of generating structurally and functionally distinct ubiquitin modifications on proteins. The complexity of protein ubiquitylation has presented significant challenges in not only identifying ubiquitylated proteins but also characterising their functional significance. Mass spectrometry enables in-depth analysis of proteins and their post-translational modification status, offering a powerful tool for studying protein ubiquitylation and its biological diversity: an approach termed ubiquitylomics. Ubiquitylomics has been employed to tackle different perspectives of ubiquitylation, including but not limited to global quantification of substrates and ubiquitin linkages, ubiquitin site recognition and crosstalk with other post-translational modifications. As the field of mass spectrometry continues to evolve, the usage of ubiquitylomics has unravelled novel insights into the regulatory mechanisms of protein ubiquitylation governing biology. However, ubiquitylomics research has predominantly been conducted in cellular models, limiting our understanding of ubiquitin signalling events driving skeletal muscle biology. By integrating the intricate landscape of protein ubiquitylation with dynamic shifts in muscle physiology, ubiquitylomics promises to not only deepen our understanding of skeletal muscle biology but also lay the foundation for developing transformative muscle-related therapeutics. This review aims to articulate how ubiquitylomics can be utilised by researchers to address different aspects of ubiquitylation signalling in skeletal muscle. We explore methods used in ubiquitylomics experiments, highlight relevant literature employing ubiquitylomics in the context of skeletal muscle and outline considerations for experimental design.

Modern technology-based exploration of mechanism of traditional Chinese medicine in prevention and treatment of gastric cancer

This review comments on the article “To explore the mechanism of Yigong San anti-gastric cancer and immune regulation”. We are interested that the article applied network pharmacology and bioinformatics techniques to elucidate the mechanism of action of Yigong Sang, a traditional Chinese medicine (TCM), in the treatment of gastric cancer (GC). The mechanism of action of Yigong Sang in the treatment of GC has not yet been elucidated because it is composed of multiple Chinese medicines with multiple components and multiple targets. The emergence of network pharmacology and bioinformatics analysis helps explain the mechanism of action of TCM in preventing and treating GC, and provides a possibility for TCM to transform from empirical to evidence-based medicine. This is of great significance for the application of TCM in oncology, new drug development, formula optimization, and the improvement of clinical efficacy.

EMS-induced missense mutation in TaCHLI-7D affects leaf color and yield-related traits in wheat.

Mutations in TaCHLI impact chlorophyll levels and yield-related traits in wheat. Natural variations in TaCHLI-7A/B influence plant productivity, offering potential for molecular breeding. Chlorophyll is essential for plant growth and productivity. The CHLI subunit of the magnesium chelatase protein plays a key role inserting magnesium into protoporphyrin IX during chlorophyll biosynthesis. Here, we identify a novel wheat mutant chlorophyll (chl) that exhibits yellow-green leaves, reduced chlorophyll levels, and increased carotenoid content, leading to an overall decline in yield-related traits. Map-based cloning reveals that the chl phenotype is caused by a point mutation (Asp186Asn) in the TaCHLI-7D gene, which encodes subunit I of magnesium chelatase. Furthermore, the three TaCHLI mutants: chl-7b-1 (Pro82Ser), chl-7b-2 (Ala291Thr), and chl-7d-1 (Gly357Glu), also showed significant reductions in chlorophyll content and yield-related traits. However, TaCHLI-7D overexpression in rice significantly decreased thousand kernel weight, yield per plant, and germination. Additionally, natural variations in TaCHLI-7A/B are significantly associated with flag leaf, spike exsertion length, and yield per plant. Notably, the favorable haplotype, TaCHLI-7B-HapII, which displayed higher thousand kernel weight and yield per plant, is positively selected in wheat breeding. Our study provides insights on the regulatory molecular mechanisms underpinning leaf color and chlorophyll biosynthesis, and highlights TaCHLI functions, which provide useful molecular markers and genetic resources for wheat breeding.

Deficiency of geranylgeranyl biphosphate synthase in kidney tubules causes cystic kidney disease.

Polycystic kidney disease (PKD) is a common hereditary kidney disease. Although PKD occurrence is associated with certain gene mutations, its onset regulatory mechanisms are still not well understood. Here, we first report that the key enzyme geranylgeranyl diphosphate synthase (GGPPS) is specifically expressed in renal tubular epithelial cells of mouse kidneys. We aimed to explore the role of GGPPS in PKD. In this study, we established a Ggppsfl/fl:Cdh16cre mouse model and compared its phenotype with that of wild-type mice. A Ggpps-downregulation HK2 cell model was also used to further determine the role of GGPPS. We found that GGPPS was specifically expressed in renal tubular epithelial cells of mouse kidneys. Its expression also increased with age. Low GGPPS expression was observed in human ADPKD tissues. In the Ggppsfl/fl:Cdh16cre mouse model, Ggpps deletion in renal tubular epithelial cells induced the occurrence and development of renal tubule cystic dilation and caused the death of mice after birth due to abnormal renal function. Enhanced proliferation of cyst-lining epithelial cells was also observed after the knockout of Ggpps. These processes were related to the increased rate of Rheb on membrane/cytoplasm and hyperactivation of mTORC1 signaling. In conclusion, the deficiency of GGPPS in kidney tubules induced the formation of renal cysts. It may play a critical role in PKD pathophysiology. A novel therapeutic strategy could be designed according to this work.

Combined analysis of lncRNA and mRNA emphasizes the potential role of tryptophan-mediated regulation of muscle development in weaned piglets by lncRNA.

Pork is an important high-value protein source that fulfills the nutritional requirements for normal growth development, repair, and metabolism. Tryptophan (Trp), a crucial amino acid for piglet growth performance and muscle development, has an essential yet unclear regulatory mechanism. To investigate the biological basis of Trp regulation of piglet muscle development and identify the related regulatory pathways, we studied 20 weaned piglets. The piglets were divided into control (CON, 0.14% Trp) and high Trp (HT, 0.35% Trp) groups. They were fed with different Trp concentrations for 28 days, after which we collected the longissimus dorsi (LD) muscle for histomorphometric analysis and RNA extraction. Our results showed that the HT diet significantly increased the average daily weight gain, myocyte number, and muscle fiber density in weaned piglets. We then analyzed the differentially expressed (DE) genes in the LD muscle through RNA sequencing (RNA-seq). We identified 253 lncRNAs and 1055 mRNAs mainly involved in myoblast proliferation and myofiber formation, particularly through the FoxO and AMPK signaling pathways and metabolism. Further analysis of the DE lncRNA targeting relationship and construction of a protein-protein interaction (PPI) network resulted in the discovery of a novel lncRNA, XLOC_021675 or FRPMD, and elucidated its role in regulating piglet muscle development. Finally, we confirmed the RNA-seq results by reverse transcription polymerase chain reaction (RT-PCR). This study provides valuable insights into the regulatory mechanism of lncRNA-mediated Trp regulation of muscle development in weaned piglets offering a theoretical basis for optimizing piglet dietary ratios and enhancing pork production.

PSIV-23 Transcriptomic analysis revealed the mechanisms of resilience and susceptibility to nonalcoholic steatohepatitis in dairy goats under high-concentrate diet

Abstract Liver health is vital for growth and health of ruminants, which can directly affect their performance. High-concentrate diet (HCD) feeding, a common practice to meet the energy requirements for animal production and growth, has been known to induce liver damage, including nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) in ruminants. To date, the regulatory mechanisms of liver metabolic dysfunctions in ruminants induced by HCD are not well defined. To elucidate molecular regulatory mechanisms underlying liver dysfunction in ruminants, we compared liver transcriptome and single-cell transcriptome of dairy goats that had varied responses to the HCD, aiming to underpin the key mechanisms behind resilience and susceptibility to NASH in ruminants. After feeding 30 dairy goats with low-concentrate diet (LCD, n = 15, defined as Con) or high-concentrate diet (HCD, n = 15) for 6 wk, 10 of them developed NASH (defined as NASH), while the other 5 did not showed any NASH symptom (defined as NASH-tolerance, NASH-T) under HCD. Liver tissues were collected to extract RNA. The paired-end RNA-seq library was sequenced with a NovaSeq6000 sequencer (Illumina). Real-time quantitative PCR was conducted to verify the significantly different genes among groups. Liver single-cell transcriptome was performed using 10×Genomics Chromium system. Liver transcriptome analysis revealed that the expression level of genes involved in the interleukin (IL)-17 signaling pathway, including IL-17A (P = 0.035; 0.001), Act1 (P = 0.036; 0.002), FOSB (P = 0.041; 0.048) and IL-1β (P = 0.022; 0.002) in the NASH group were significantly greater than those in the Con and NASH-T groups. Hepatic single-cell RNA sequencing identified seven cell types in the liver, including hepatocytes, macrophages, T cells, endothelial cell, natural killer (NK) cell, hepatic stellate cells (HSCs), B cells. In T cell subset, significantly decreased T helper 17 (TH17) cells and increased regulatory T (Treg) cells were detected in the NASH group compared with Con and NASH-T groups. In TH17 cell, the expression levels of IL-17A (P = 0.004; 0.009) and IL-23R (P = 0.007; 0.01; the surface antibodies of TH17 cells) in NASH group were significantly greater than those in the Con and NASH-T groups. The expression levels of Foxp3 (P < 0.001; 0.013), IL-2R (P < 0.001; 0.001), CTLA-4 (P = 0.005; 0.017) and CD127 (P = 0.004; 0.009; the surface antibodies of Treg cells) in NASH group were significantly less than those in the Con and NASH-T groups. Our results revealed the role of liver immune cells in maintaining tolerance to NASH in dairy goats under HCD, which provides new understanding in pathogenesis of NASH under HCD. Further studies are needed to identify major factors that drive the changes of ratio of TH17/Treg cell.

Acetylomics reveals an extensive acetylation diversity within Pseudomonas aeruginosa

Abstract Bacteria employ a myriad of regulatory mechanisms to adapt to the continuously changing environments that they face. They can, for example, use post-translational modifications, such as Nε-lysine acetylation, to alter enzyme activity. Although a lot of progress has been made, the extent and role of lysine acetylation in many bacterial strains remains uncharted. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) in combination with the immunoprecipitation of acetylated peptides and LC-MS/MS to measure the first Pseudomonas aeruginosa PAO1 acetylome, revealing 1 076 unique acetylation sites in 508 proteins. Next, we assessed inter-strain acetylome differences within P. aeruginosa by comparing our PAO1 acetylome with two publicly available PA14 acetylomes, and postulate that the overall acetylation patterns are not driven by strain-specific factors. In addition, the comparison of the P. aeruginosa acetylome to 30 other bacterial acetylomes revealed that a high percentage of transcription related proteins are acetylated in the majority of bacterial species. This conservation could help prioritize the characterization of functional consequences of individual acetylation sites.

Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

BiOBr/FeMoO4 composite achieves oxygen vacancy concentration adjustment to promote persulfate activation degradation of organic pollutants in saline water

The investigation about the mechanism of crystal plane regulation on the generation of oxygen vacancies remains a challenge. In this paper, BiOBr/FeMoO4 composites were synthesized by precise control of crystal plane growth, and it exhibited the enhanced concentration of oxygen vacancies due to lower formation energy of oxygen vacancies. The composite performs higher photo-Fenton-like ability for degrading oxytetracycline hydrochloride (OTC). Structural analyses and theoretical calculations reveal that crystal plane regulation induces significant changes in oxygen vacancy concentration. The BiOBr/FeMoO4/peroxydisulphate (PDS) /light system, which dominated by the non-radical pathway, degraded 96.8 % ± 1.0 % of OTC within 30 min. The activation mechanism of the system and the degradation pathway of OTC were elucidated. The intermediates in the degradation process of OTC were evaluated using liquid chromatograph-mass spectrometer (LC-MS), toxicity evaluation software tool (T.E.S.T) and soybean germination experiments. This work offers novel insights into the pivotal role of crystal plane directional regulation in the quantitative generation of oxygen vacancies.

An intrinsic mechanism of metabolic tuning promotes cardiac resilience to stress.

Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress.

Exploring the roles of SPARC as a proinflammatory factor and its potential as a novel therapeutic target against cardiovascular disease.

Cardiovascular disease (CVD) is a leading cause of death worldwide, and the number of CVD patients continues to increase despite extensive research and developments in this field. Chronic inflammation is a pivotal pathological component of CVD, and unveiling new proinflammatory factors will help devise novel preventive and therapeutic strategies. The extracellular matrix (ECM) not only provides structural support between cells, but also contributes to cellular functions. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein that is particularly induced during development and tissue remodeling. A proinflammatory role for SPARC has been demonstrated in various animal models, such as in the lipopolysaccharide-induced footpad model and dextran sodium sulfate-induced colitis model. Recent clinical studies reported a positive correlation between elevated plasma SPARC levels and hypertension, obesity, and the inflammatory marker high-sensitive C-reactive protein. In addition, SPARC gene deletion attenuates the cardiac injury induced by aging, myocardial infarction, and pressure-load, suggesting that SPARC has deleterious effects on CVD. This review summarizes the regulatory and proinflammatory mechanisms of SPARC on CVD, chronic kidney disease (CKD) and cerebrovascular disease, and discusses the rationale behind measuring SPARC as a biomarker of CVD, and the effects of inhibition of SPARC in the prevention and treatment of CVD.

Autoregulated splicing of TRA2β programs T cell fate in response to antigen-receptor stimulation.

T cell receptor (TCR) sensitivity to peptide-major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2β in mouse and human. TRA2β-PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2β-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2β-PE reinclusion allowed T cell survival. Finally, we found that TRA2β-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2β-PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.

ING5 inhibits aerobic glycolysis of lung cancer cells by promoting TIE1-mediated phosphorylation of pyruvate dehydrogenase kinase 1 at Y163.

Aerobic glycolysis is critical for tumor growth and metastasis. Previously, we have found that the overexpression of the inhibitor of growth 5 (ING5) inhibits lung cancer aggressiveness and epithelial-mesenchymal transition (EMT). However, whether ING5 regulates lung cancer metabolism reprogramming remains unknown. Here, by quantitative proteomics, we showed that ING5 differentially regulates protein phosphorylation and identified a new site (Y163) of the key glycolytic enzyme PDK1 whose phosphorylation was upregulated 13.847-fold. By clinical study, decreased p-PDK1Y163 was observed in lung cancer tissues and correlated with poor survival. p-PDK1Y163 represents the negative regulatory mechanism of PDK1 by causing PDHA1 dephosphorylation and activation, leading to switching from glycolysis to oxidative phosphorylation, with increasing oxygen consumption and decreasing lactate production. These effects could be impaired by PDK1Y163F mutation, which also impaired the inhibitory effects of ING5 on cancer cell EMT and invasiveness. Mouse xenograft models confirmed the indispensable role of p-PDK1Y163 in ING5-inhibited tumor growth and metastasis. By siRNA screening, ING5-upregulated TIE1 was identified as the upstream tyrosine protein kinase targeting PDK1Y163. TIE1 knockdown induced the dephosphorylation of PDK1Y163 and increased the migration and invasion of lung cancer cells. Collectively, ING5 overexpression-upregulated TIE1 phosphorylates PDK1Y163, which is critical for the inhibition of aerobic glycolysis and invasiveness of lung cancer cells.

Greater variation of soil organic carbon in limestone- than shale-based soil along soil depth in a subtropical coniferous forest within a karst faulted basin of China

Lithology strongly influences soil microbial traits and edaphic factors and in turn soil organic carbon (SOC) dynamics. However, the effect of lithology on microbial traits, edaphic factors and resulting SOC physical fractions variation along soil depth remains inadequately understood in karst faulted basin of China. This understanding is critical for improving SOC stability. By separating SOC into labile particulate organic carbon (POC) and stable mineral-associated organic carbon (MAOC) over karst limestone and non-karst shale soil in a subtropical coniferous forest, we aimed to assess potential regulatory mechanisms underlying lithology-associated SOC stability variations across soil depth by integrating soil nutrients, mineralogical characteristics, and microbial traits. We found that SOC and its fractions were higher in limestone than in shale soil, which implying vegetation restoration effects on SOC and its fractions partly depending on lithology. Additionally, we found that the effects of soil depth on SOC and its fractions were greater in limestone soils than shale soils, and the ratios of MAOC to SOC (MAOC:SOC) and MAOC to POC (MAOC:POC) show a opposite trend in response to soil depth between two the lithologies. Variation partitioning and random forest analyses revealed that among multiple factors, the variation of SOC stability assessed via MAOC:SOC was mainly explained by microbial traits than soil nutrients and mineral properties. Contrast to soil depth, structural equation modeling analyses showed that lithology was the primary factor controlling the SOC stability when microbial traits, soil nutrients and mineralogical characteristics were controlled as conditional variables. Overall, these results highlight the crucial role of lithology in regulating the SOC stability along soil depth, which improve our understanding and management of soil carbon (C) pool in karst faulted basin of southwest China.

Pepper RING-Type E3 Ligase CaFIRF1 Negatively Regulates the Protein Stability of Pepper Stress-Associated Protein, CaSAP14, in the Dehydration Stress Response.

As part of the cellular stress response in plants, the ubiquitin-proteasome system (UPS) plays a crucial role in regulating the protein stability of stress-related transcription factors. Previous study has indicated that CaSAP14 is functionally involved in enhancing pepper plant tolerance to dehydration stress by modulating the expression of downstream genes. However, the comprehensive regulatory mechanism underlying CaSAP14 remains incompletely understood. Here, we identified a RING-type E3 ligase, CaFIRF1, which interacts with and ubiquitinates CaSAP14. Pepper plants with silenced CaFIRF1 exhibited a dehydration-tolerant phenotype when subjected to dehydration stress, while overexpression of CaFIRF1 in pepper and Arabidopsis resulted in reduced dehydration tolerance. Co-silencing of CaFIRF1 and CaSAP14 in pepper increased sensitivity to dehydration, suggesting that CaFIRF1 acts upstream of CaSAP14. A cell-free degradation analysis demonstrated that silencing of CaFIRF1 led to decreased CaSAP14 protein degradation, implicating CaFIRF1 in the regulation of CaSAP14 protein via the 26S proteasomal degradation pathway. Our findings suggest a mechanism by which CaFIRF1 mediates the ubiquitin-dependent proteasomal degradation of CaSAP14, thereby influencing the response of pepper plants to dehydration stress.

STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A

BackgroundCholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified.MethodsWe confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model.ResultsThis study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration.ConclusionsThese findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.Graphical

A 3’UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3’UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3’UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.