Dephosphorylation Activity Research Articles

Guide DNA dephosphorylation-modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B1

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids. To lend PfAgo toolbox to extended non-nucleic acid detection, we systematically investigated the mechanism characteristic of PfAgo′ preference for guide DNA (gDNA) and proposed a gDNA dephosphorylation-modulated PfAgo sensor for the detection of non-nucleic acid targets. Our results indicated that PfAgo exhibits preference for 5′-phosphorylated gDNA at a specific ratio of PfAgo to gDNA concentration. Leveraging this PfAgo′ preference and the dephosphorylation activity of alkaline phosphatase (ALP), ALP could be detected as low as 2.7 U/L. Furthermore, the PfAgo was coupled with immunolabelled ALP to develop a PfAgo-based fluorescence immunosensor, which achieves aflatoxins B1 detection with a detection limit of 29.89 pg/mL and exhibits satisfactory recoveries in wheat and maize samples. The developed method broadens the application scope of PfAgo toolbox, and provides a simple, sensitive, and universal detection platform for a variety targets.

Erdafitinib promotes ferroptosis in human uveal melanoma by inducing ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling axis

Uveal melanoma (UM) is a rare yet lethal primary intraocular malignancy affecting adults. Analysis of data from The Cancer Genome Atlas (TCGA) database revealed that FGFR1 expression was increased in UM tumor tissues and was linked to aggressive behavior and a poor prognosis. This study assessed the anti-tumor effects of Erdafitinib, a selective pan-FGFR inhibitor, in both in vitro and in vivo UM models. Erdafitinib exhibited a robust anti-cancer activity in UM through inducing ferroptosis in the FGFR1-dependent manner. Transcriptomic data revealed that Erdafitinib mediated its anti-cancer effects via modulating the ferritinophagy/lysosome biogenesis. Subsequent research revealed that Erdafitinib exerted its effects by reducing the expression of FGFR1 and inhibiting the activity of mTORC1 in UM cells. Concurrently, it enhanced the dephosphorylation, nuclear translocation, and transcriptional activity of TFEB. The aggregation of TFEB in nucleus triggered FTH1-dependent ferritinophagy, leading to lysosomal activation and iron overload. Conversely, the overexpression of FGFR1 served to mitigate the effects of Erdafitinib on ferritinophagy, lysosome biogenesis, and the activation of the mTORC1/TFEB signaling pathway. In vivo experiments have convincingly shown that Erdafitinib markedly curtails tumor growth in an UM xenograft mouse model, an effect that is closely correlated with a decrease in FGFR1 expression levels. The present study is the first to demonstrate that Erdafitinib powerfully induces ferroptosis in UM by orchestrating the ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling. Consequently, Erdafitinib emerges as a strong candidate for clinical trial investigation, and FGFR1 emerges as a novel and promising therapeutic target in the treatment of UM.

Dual-specificity phosphatase 26 protects against kidney injury caused by ischaemia-reperfusion through restraint of TAK1-JNK/p38-mediated apoptosis and inflammation of renal tubular epithelial cells

Dual-specificity phosphatase 26 (DUSP26) acts as a pivotal player in the transduction of signalling cascades with its dephosphorylating activity. Currently, DUSP26 attracts extensive attention due to its particular function in several pathological conditions. However, whether DUSP26 plays a role in kidney ischaemia-reperfusion (IR) injury is unknown. Aims of the current work were to explore the relevance of DUSP26 in kidney IR damage. DUSP26 levels were found to be decreased in renal tubular epithelial cells following hypoxia-reoxygenation (HR) and kidney samples subjected to IR treatments. DUSP26-overexpressed renal tubular epithelial cells exhibited protection against HR-caused apoptosis and inflammation, while DUSP26-depleted renal tubular epithelial cells were more sensitive to HR damage. Upregulation of DUSP26 in rat kidneys by infecting adenovirus expressing DUSP26 markedly ameliorated kidney injury caused by IR, while also effectively reducing apoptosis and inflammation. The mechanistic studies showed that the activation of transforming growth factor-β-activated kinase 1 (TAK1)-JNK/p38 MAPK, contributing to kidney injury under HR or IR conditions, was restrained by increasing DUSP26 expression. Pharmacological restraint of TAK1 markedly diminished DUSP26-depletion-exacebated effects on JNK/p38 activation and HR injury of renal tubular cells. The work reported a renal-protective function of DUSP26, which protects against IR-related kidney damage via the intervention effects on the TAK1-JNK/p38 axis. The findings laid a foundation for understanding the molecular pathogenesis of kidney IR injury and provide a prospective target for treating this condition.

AMBRA1 promotes intestinal inflammation by antagonizing PP4R1/PP4c mediated IKK dephosphorylation in an autophagy-independent manner.

IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

Effect of Surface Treatment of Nanocrystalline CeO2 on Its Dephosphorylation Activity and Adsorption of Inorganic Phosphates.

The surface of nanocrystalline cerium oxide (CeO2) was treated with various chemical agents by a simple postmodification method at 25 °C and atmospheric pressure. Hydrogen peroxide, ammonium persulfate, deionized water, ascorbic acid, and ortho-phosphoric acid were used in order to study and evaluate their effect on surface materials, such as surface area, crystallite size, number of surface hydroxyl groups, particle morphology, and Ce3+/Ce4+ ratio. Paraoxon-methyl (PO) decomposition and inorganic phosphate adsorption were used to evaluate the effect of surface treatment on catalytic and adsorption properties. CeO2 surface was studied by Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and acid-base titration. While the treatment procedure affected the number of surface hydroxyl groups and the amount of bulk surface oxygen vacancies, only negligible changes were observed in the Ce3+/Ce4+ ratio. Interestingly, surface treatment affected the ability to decompose PO, but only a small effect on inorganic phosphate adsorption was observed, indicating the robustness of CeO2 for the latter. A mechanism for possible interaction of the used chemicals with the CeO2 surface was proposed.

Harmine and Kaempferol treatment enhances NFATC1 and FOXP3 mediated regulatory T-cells’ suppressive capacity in generalized vitiligo

BackgroundGeneralized vitiligo (GV) is an autoimmune disease characterized by the progressive loss of melanocytes. ObjectivesCurrent study was undertaken to assess in-vitro therapeutic potential of Harmine and Kaempferol for GV. MethodsCalcium, calcineurin, NFATC1 levels, cell proliferation were assessed by various kits and ORAI1, PEIZO1, Calcineurin, GSK3B, DYRK1A transcripts and IFN-γ,IL-10,TGF-β protein levels were assessed by qPCR and ELISA in blood and skin biopsy samples from Tregs of 52 patients and 50 controls. ResultsHarmine and Kaempferol treatment enhances Treg suppressive capacity, NFATs and FOXP3 expression in blood and skin Tregs of GV patients (p < 0.05). Furthermore, Harmine and Kaempferol treatment in Tregs increased calcineurin and NFATC1 activity and decreased DYRK1A transcripts in blood and skin Tregs of GV patients(p < 0.05). In-silico analysis revealed that Harmine and Kaempferol might boost Treg suppressive capacity by increasing calcineurin dephosphorylation activity leading to increase NFATs activation and also increase nuclear retention of NFATs by inhibiting DYRK1a phosphorylation activity. Moreover, calcineurin and NFATC1 activity in Tregs were positively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05), whereas, DYRK1A transcripts were negatively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05). These compounds significantly increased melanocytes’ survival and proliferation in Treg:CD4+/CD8+:SK-Mel-28 cell line co-culture system from GV patients (p < 0.0001). ConclusionsFor the first time the study suggests that Harmine and Kaempferol treated Tregs could control the CD8+ and CD4+T-cells’ proliferation and IFN-γ production, leading to melanocytes’ survival and proliferation. These compounds may serve as novel Treg-based therapeutics for GV; however, in vivo studies are warranted to assess the safety and efficacy of these compounds.

Effect of Phosphorylation Sites Mutations on the Subcellular Localization and Activity of AGPase Bt2 Subunit: Implications for Improved Starch Biosynthesis in Maize

ADP-Glc pyrophosphorylase (AGPase) is a pivotal enzyme catalyzing the conversion of ATP and glucose-1-phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc), thereby serving as a rate-limiting factor in starch biosynthesis in crops. Although previous investigations have suggested phosphorylation-based regulation of AGPase in maize, the explicit modulation mechanisms have yet to be elucidated. This research evaluated the effect of point mutations at phosphorylation sites (identified using iTRAQTM AB SCIEX, Framingham, MA, USA) on the subcellular localization and activity of the AGPase small subunit Bt2, and its interaction with the large subunit Sh2, in maize. Despite the induction of point mutations, subcellular localization of the Bt2 subunit remained unaltered, primarily within the cytoplasm and nucleus. The interaction between Bt2 and Sh2 subunits continued, mainly in the chloroplast. Notably, an increase in AGPase activity was observed in the case of simulated phosphorylation point mutations, whereas dephosphorylation activity significantly diminished relative to the wild type. These findings demonstrate that point mutations do not affect the subcellular localization of the Bt2 subunit or its interaction with the Sh2 subunit, but substantially modulate AGPase activity. This study provides critical insights into the role of point mutations in enhancing AGPase activity, thus potentially accelerating the production of ADP-Glc, the primary substrate for starch synthesis, promising implications for improved starch biosynthesis in maize.

Longitudinal genome-wide association analysis using a single-step random regression model for height in Japanese Holstein cattle

Growth traits, such as body weight and height, are essential in the design of genetic improvement programs of dairy cattle due to their relationship with feeding efficiency, longevity, and health. We investigated genomic regions influencing height across growth stages in Japanese Holstein cattle using a single-step random regression model. We used 72,921 records from birth to 60 mo of age with 4,111 animals born between 2000 and 2016. The analysis included 1,410 genotyped animals with 35,319 single nucleotide polymorphisms, consisting of 883 females with records and 527 bulls, and 30,745 animals with pedigree information. A single genomic region at the 58.4 megabase pair on chromosome 18 was consistently identified across 6 age points of 10, 20, 30, 40, 50, and 60 mo after multiple testing corrections for the significance threshold. Twelve candidate genes, previously reported for longevity and gestation length, were found near the identified genomic region. Another location near the identified region was also previously associated with body conformation, fertility, and calving difficulty. Functional Gene Ontology enrichment analysis suggested that the candidate genes regulate dephosphorylation and phosphatase activity. Our findings show that further study of the identified candidate genes will contribute to a better understanding of the genetic basis of height in Japanese Holstein cattle.

ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves.

5-Aminolevulinic acid (ALA), known as a new natural plant growth regulator, can reverse abscisic acid (ABA)-induced stomatal closure. The protein phosphatase 2A (PP2A) played an important role in regulation of stomatal movement by ALA and ABA; however, the underlying molecular mechanisms remain unclear. Here, we report that ALA promotes MdPP2A activity and gene expression in the leaf epidermis of apple (Malus × domestica Borkh.), and expression of the catalytic subunit MdPP2AC was most significantly correlated with stomatal aperture. Western blotting showed that ALA enhanced MdPP2AC protein abundance and phosphorylation. Y2H (yeast two hybrid), FLC (firefly luciferase complementation imaging) and BiFC (Bimolecular fluorescence complementation) assays showed that MdPP2AC interacted with several other MdPP2A subunits as well as MdSnRK2.6 (Sucrose non-fermenting 1-related protein kinase 2.6), and the latter interaction was further verified by pull-down and MST (microscale thermophoresis) assays. ALA downregulated ABA-induced MdSnRK2.6 gene expression, kinase activity, and protein phosphorylation. In transiently transgenic apple leaves, OE-MdPP2AC promoted stomatal aperture by reducing Ca2+ and H2O2 levels but increasing flavonol levels in guard cells. Conversely, OE-MdSnRK2.6 induced stomatal closure by increasing Ca2+ and H2O2 but reducing flavonols. Partial silencing of these genes had opposite effects on Ca2+, H2O2, flavonols, and stomatal movement. Application of exogenous ALA stimulated PP2A activity, which promoted SnRK2.6 dephosphorylation and lower kinase activity in wild-type and transgenic apple leaves. We therefore propose that PP2AC, which dephosphorylates SnRK2.6 and represses its enzyme activity, mediates ALA signaling to inhibit ABA-induced stomatal closure in apple leaves.

Identification of linderalactone as a natural inhibitor of SHP2 to ameliorate CCl4-induced liver fibrosis.

Liver fibrosis is characterised by the activation of hepatic stellate cells (HSCs) and matrix deposition. Accumulating evidence has revealed that the oncogenic protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) acts as a therapeutic target of fibrosis. Although several SHP2 inhibitors have reached early clinical trials, there are currently no FDA-approved drugs that target SHP2. In this study, we aimed to identify novel SHP2 inhibitors from an in-house natural product library to treat liver fibrosis. Out of the screened 800 compounds, a furanogermacrane sesquiterpene, linderalactone (LIN), significantly inhibited SHP2 dephosphorylation activity in vitro. Cross-validated enzymatic assays, bio-layer interferometry (BLI) assays, and site-directed mutagenesis were used to confirm that LIN directly binds to the catalytic PTP domain of SHP2. In vivo administration of LIN significantly ameliorated carbon tetrachloride (CCl4)-induced HSC activation and liver fibrosis by inhibiting the TGFβ/Smad3 pathway. Thus, LIN or its derivatives could be considered potential therapeutic agents against SHP2-related diseases, such as liver fibrosis or NASH.

Exogenous c-di-GMP inhibited the biofilm formation of Vibrio splendidus.

Vibrio splendidus, a gram-negative bacterium that is ubiquitously present in marine environments, has been increasingly deemed an important opportunistic pathogen of marine animals. In this study, the biofilm formation of V. splendidus was quantitatively determined and morphologically characterized. Three stages of biofilm formation, including adhesion, aggregation and maturation were observed in the biofilm formed by V. splendidus. The inhibitory effect of exogenous bis (3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) on the biofilm formation from the scratch and preformed established biofilms of V. splendidus was determined. When 200μmol/L c-di-GMP was added, the quantity of biofilm decreased by 88.1% or 66.7% under the two conditions. To explore the preliminary mechanism of exogenous c-di-GMP on the biofilm formed by V. splendidus, proteomic analysis was performed. GO enrichment analysis showed that exogenous c-di-GMP upregulated biological processes, including the tricarboxylic acid cycle, oxidation‒reduction reactions and organonitrogen compound catabolism and significantly downregulated tRNA threonylcarbamoyladenosine modification, protein dephosphorylation, and lactate transmembrane transporter activity. Sequence-specific DNA binding activity was the most markedly downregulated molecular function. KEGG analysis showed that the valine, leucine and isoleucine degradation pathway was the most enriched pathway, followed by nitrogen metabolism, among the 20 upregulated pathways. Among the downregulated pathways, a nonribosomal peptide structure pathway and the streptomycine, polyketide sugar unit, acarbose and validamycin biosynthesis pathways were significantly enriched. Our present study provides basic data for the biofilm formation of V. splendidus and the preliminary inhibitory mechanism of exogenous c-di-GMP on the biofilm formation of V. splendidus.

Characterization of the active response of a guinea pig carotid artery.

This work presents a characterization of the active response of the carotid artery of guinea pig fetuses through a methodology that encompasses experiments, modeling and numerical simulation. To this end, the isometric contraction test is carried out in ring samples subjected to different levels of KCl concentrations and pre-stretching. Then, a coupled mechanochemical model, aimed at describing the smooth cell behavior and its influence on the passive and active mechanical response of the vascular tissue, is calibrated from the experimental measurements. Due to the complex stress and strain fields developed in the artery, a finite element numerical simulation of the test is performed to fit the model parameters, where those related to the phosphorylation and dephosphorylation activity along with the load-bearing capacity of the myosin cross-bridges are found to be the most predominant when sensitizing the active response. The main strengths of the model are associated with the prediction of the stationary state of the active mechanical response of the tissue through a realistic description of the mechanochemical process carried out at its cellular level.

Immunosuppressive Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

Three new cytochalasins, phomoparagins A-C (1–3), along with five known analogs (4–8), were isolated from Phomopsis asparagi DHS-48, a mangrove-derived endophytic fungus. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD techniques. Notably, 1 possessed an unprecedented 5/6/5/8/5-fused pentacyclic skeleton. These compounds were tested for their inhibitory activity against concanavalin A (ConA)/lipopolysaccharide (LPS)-induced spleen lymphocyte proliferation and calcineurin (CN) enzyme. Several metabolites (2 and 4–6) exhibited fascinating inhibitory activities with a relatively low toxicity. Furthermore, 2 was demonstrated to inhibit ConA-stimulated activation of NFAT1 dephosphorylation and block NFAT1 translocation in vitro, subsequently inhibiting the transcription of interleukin-2 (IL-2). Our results provide evidence that 2 may, at least partially, suppress the activation of spleen lymphocytes via the CN/NFAT signaling pathway, highlighting that it could serve as an effective immunosuppressant that is noncytotoxic and natural.

A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.

Towards discovery of inhibitors of the undecaprenyl-pyrophosphate phosphatase BacA by virtual high-throughput screening

Increasing resistance to common antibiotics is becoming a major challenge that requires the development of new antibacterial agents. Peptidoglycan is an essential heteropolymer of the bacterial envelope that ensures the integrity and shape of all bacteria and is also an important target for antibiotics. The biosynthesis of peptidoglycan depends on a lipid carrier, undecaprenyl phosphate. As a byproduct of peptidoglycan polymerization, the lipid carrier is released as undecaprenyl pyrophosphate, which must be recycled to allow new polymerization cycles. To this end, it undergoes a dephosphorylation process catalyzed by the membrane phosphatase BacA, which is specific and highly conserved in bacteria. In the present study, we identified small molecules displaying inhibitory potency towards BacA. We began by preparing a commercial compound library, followed by high-throughput virtual screening by ensemble docking using the 3D structure of BacA and molecular dynamics snapshots to account for the flexibility of the protein. Of 83 compounds computationally selected and tested in a biochemical assay, one sulfamoylthiophene molecule showed significant inhibition of the undecaprenyl pyrophosphate dephosphorylation activity catalyzed by BacA. Subsequently, an additional 33 scaffold analogs were selected and acquired, of which 6 compounds exhibited BacA inhibition. The IC50 values of these compounds ranged from 42 to 366 μM. In addition, significant antibacterial activity against Escherichia coli was observed in TolC/PAP2-depleted strains. We believe that the overall strategy followed in this study and the identified class of inhibitors provide a solid foundation for the further development of potent BacA-targeted inhibitors and the discovery of novel antibacterial compounds.

Role of active site arginine residues in substrate recognition by PPM1A

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.

Adaptations in Hippo-Yap signaling and myofibroblast fate underlie scar-free ear appendage wound healing in spiny mice.

Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments invitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys invivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-β1-induced human MF formation invitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing invivo.

Regulation PP2Ac methylation ameliorating autophagy dysfunction caused by Mn is associated with mTORC1/ULK1 pathway

Manganese (Mn) exposure leads to autophagy dysfunction and causes neurodegenerative diseases such as Parkinson's syndrome and Alzheimer's disease. However, the mechanism of neurotoxicity of Mn has been less clear. The methylation of the protein phosphatase 2A catalytic subunit determines the dephosphorylation activity of protein phosphatase and plays an important role in autophagy regulation. In this investigation, we established a model of Mn (0–2000 μmol/L) exposure to N2a cells for 12 h, used the PPME-1 inhibitor ABL-127, and constructed an LCMT1-overexpressing N2a cell line. We also regulated the PP2Ac methylation level and explored the effect of PP2Ac methylation on Mn-induced (0–1000 μmol/L) N2a cellular autophagy. Our results showed that Mn > 500 μmol/L induced N2a cell damage and increased oxidative stress. Moreover, Mn modulated autophagy in N2a cells by downregulating PP2Ac methylation, which regulated mTORC1 signaling pathway activation. Both ABL-127 and LCMT1 overexpression can upregulate PP2Ac methylation in parallel with ameliorating N2a cell abnormal autophagy induced by Mn, Briefly, the upregulation of PP2Ac methylation can ameliorate the autophagy disorder of N2a by Mn and effectively alleviate Mn-induced cytotoxicity and oxidative stress, indicating that regulation of autophagy is a protective strategy against Mn-induced neurotoxicity.

Abstract PR12: Paracrine orchestration of intestinal tumorigenesis at the mesenchymal-epithelial interface

Abstract Intestinal tumor initiation is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types. It remains unknown whether the physiologic mesenchymal microenvironment of mutant stem cells affects tumor initiation. Here we show that the mesenchymal niche controls tumor initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme by single cell RNA-seq, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts (RPPFs) that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumor initiation in two different models of sporadic, autochthonous tumorigenesis. At the mechanistic level, by performing single-cell RNA-seq analyses of a mesenchymal niche organotypic model, we found that fibroblast-derived PGE2 reprograms intestinal stem cells into Tuft cell precursors. These are characterized by the activation of a strong tumor initiation program driven by the Hippo pathway effector Yap, a molecule indispensable for intestinal tumorigenesis. Organoid experiments established a novel molecular mechanism whereby PGE2 activates Yap dephosphorylation, nuclear translocation, and transcriptional activity by signaling specifically via receptor Ptger4. In vivo, epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells, abrogated Yap target gene activation, and prevented sporadic tumor initiation in mutant mice, thereby demonstrating the robust paracrine control of tumor-initiating stem cells by PGE2/Ptger4. Patient-derived organoid analyses established that PGE2/ PTGER4 also regulate stem cell function in humans. Our study demonstrates for the first time that colorectal cancer initiation is orchestrated by the mesenchymal niche. Moreover, it reveals a mechanism whereby RPPFs exert paracrine control over tumor-initiating stem cells via the druggable PGE2/Ptger4/Yap signaling axis. Citation Format: Manolis Roulis, Emilios Kaklamanos, George Kollias, Richard Flavell. Paracrine orchestration of intestinal tumorigenesis at the mesenchymal-epithelial interface [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR12.

Xanthomonas axonopodis pv. punicae depends on multiple non-TAL (Xop) T3SS effectors for its coveted growth inside the pomegranate plant through repressing the immune responses during bacterial blight development

Xanthomonas axonopodis pv. punicae (Xap), the bacterial blight pathogen of pomegranate, incurs substantial loss to yield and reduces export quality of this economically important fruit crop. During infection, the bacterium secretes six non-TAL (Xop) effectors into the pomegranate cells through a specialized type three secretion system (T3SS). Previously, we demonstrated the role of two key effectors, XopL and XopN in pathogenesis. Here, we investigate the role of rest effectors (XopC2, XopE1, XopQ and XopZ) on disease development. We generated null mutants for each individual effector and mutant bacterial suspension was infiltrated into pomegranate leaves. Compared to Xap wild, the mutant bacterial growth was reduced by 2.7–11.5 folds. The mutants produced lesser water-soaked lesions when infiltrated on leaves by 1.13–2.21 folds. Among the four effectors, XopC2 contributes highest for in planta bacterial growth and disease development. XopC2 efficiently suppressed the defense responses like callose deposition, reactive oxygen species (ROS) and the activation of immune responsive genes. Being a major contributor, we further characterize XopC2 for its subcellular localization, its protein structure and networking. XopC2 is localized to the plasma membrane of Nicotiana benthamiana like XopL and XopN. XopC2 is a 661 amino acids protein having 15 alpha and 17 beta helix. Our STRING and I-TASSER based analysis hinted that XopC2 interacts with multiple membrane localized plant proteins including transcription regulator of CCR4-NOT family, TTN of maintenance of chromosome family and serine/threonine-protein phosphatase 2A (PP2A) isoform. Based on the interaction it is predicted that XopC2 might involve in diverse functions like nuclear-transcribed mRNA catabolic process, maintenance of chromosome, hormone signaling and protein dephosphorylation activities and thereby suppress the plant immunity. Altogether, our study suggests that Xap largely depends on three non-TAL (Xop) effectors, including XopC2, XopL and XopN, to modulate pomegranate PTI for its unrestricted proliferation during bacterial blight development.