Search Results (26)

The secondary contamination of nodularin disinfection by-products (NOD-DBPs) is a problem worthy of attention. In this study, prototypical NOD-R-DBPs were prepared, and their toxicity was assessed using conventional protein phosphatase (PPs) inhibition assay, confirming that structural changes in “Adda³” during chlorination are key factors leading to a significant reduction in NOD-R toxicity. However, some NOD-R-DBPs still exhibit certain levels of toxicity (2.8–81% of NOD-R). To elucidate the mechanism underlying the potential inhibitory effect of NOD-R-DBPs on protein phosphatase 2A (PP2A), molecular simulations were employed to establish interaction models between prototypical NOD-R-DBPs and PP2A using homology modeling strategies, and molecular docking was used to obtain candidate interaction parameters between prototypical NOD-R-DBPs and PP2A. Structural changes in “Adda³” weakened the hydrogen bonds “Adda³”Asn₁₁₇ and “Adda³”His₁₁₈. Subsequently, the disruption of “Adda³” altered key interactions between NOD-R-DBPs and PP2A (hydrogen bond Mdhb⁵ ← Arg₈₉, ionic bond Glu⁴-Arg₈₉, metal bond His₂₄₁-Mn₁²⁺, etc.). The changes in these interactions further altered the interactions between conserved amino acids and the catalytic center Mn²⁺ (ionic bond Asp₅₇-Mn₂²⁺), thereby increasing Mn²⁺ exposure. Meanwhile, the retained interactions promoted the binding of -PO₄ with the conserved amino acids His₁₁₈ and Arg₈₉. Prototypical NOD-R-DBPs retained the aforementioned key interactions and thus exhibit potential inhibitory effects on PP2A. The varying degrees of damage to the Adda³ structure led to significant differences in the inhibitory effects of different NOD-R-DBPs on PP2A. Full article

Myosin phosphatase (MP) holoenzyme consists of protein phosphatase-1 (PP1) catalytic subunit (PP1c) associated with myosin phosphatase target subunit-1 (MYPT1) and it plays an important role in mediating the phosphorylation of the 20 kDa light chain (MLC20) of myosin, thereby regulating cell contractility. The association of MYPT1 with PP1c increases the phosphatase activity toward myosin; therefore, disrupting/dissociating this interaction may result in inhibition of the dephosphorylation of myosin. In this study, we probed how MYPT1^32–58 peptide including major PP1c interactive regions coupled with biotin and cell-penetrating TAT sequence (biotin-TAT-MYPT1) may influence MP activity. Biotin-TAT-MYPT1 inhibited the activity of MP holoenzyme and affinity chromatography as well as surface plasmon resonance (SPR) binding studies established its stable association with PP1c. Biotin-TAT-MYPT1 competed for binding to PP1c with immobilized GST-MYPT1 in SPR assays and it partially relieved PP1c inhibition by thiophosphorylated (on Thr696 and Thr853) MYPT1. Moreover, biotin-TAT-MYPT1 dissociated PP1c from immunoprecipitated PP1c-MYPT1 complex implying its holoenzyme disrupting ability. Biotin-TAT-MYPT1 penetrated into A7r5 smooth muscle cells localized in the cytoplasm and nucleus and exerted inhibition on MP with a parallel increase in MLC20 phosphorylation. Our results imply that the biotin-TAT-MYPT1 peptide may serve as a specific MP regulatory cell-penetrating peptide as well as possibly being applicable to further development for pharmacological interventions. Full article

Type 1 protein phosphatases (PP1s) are crucial in various plant cellular processes. Their function is controlled by regulators known as PP1-interacting proteins (PIPs), often intrinsically disordered, such as Inhibitor 2 (I2), conserved across kingdoms. The durum wheat TdRL1 acts as a positive regulator of plant stress tolerance, presumably by inhibiting PP1 activity. In this work, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrate that the durum wheat TdPP1 interacts with both TdRL1 and At-I2 in vivo. YFP fluorescence restored after TdRL1-TdPP1 interaction decorated specifically the microtubular network of the tobacco co-infiltrated cells. In vitro phosphatase assays revealed that TdRL1 inhibited the activity of wild-type TdPP1 and two mutant forms (T243M and H135A) in a concentration-dependent manner, showing a novel and potent inhibition mechanism. Structural modeling of the TdPP1-inhibitor complexes suggested that both At-I2 and TdRL1 bind to TdPP1 by wrapping their flexible C-terminal tails around it, blocking access to the active site. Remarkably, the model showed that TdRL1 differs from At-I2 in its interaction with TdPP1 by trapping the phosphatase with its N-terminal tail. These findings provide important insights into the regulatory mechanisms governing the activity of PP1s in plants and highlight the potential for targeted inhibition to modulate plant stress responses. Full article

Okadaic acids (OAs) are causative agents of diarrhetic shellfish poisoning, produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp. Microcystins (MCs) are cyclic heptapeptide hepatotoxins produced by some cyanobacteria genera, including Microcystis spp. Traditionally, toxicity detection and quantification of these natural toxins were performed using a mouse bioassay (MBA); however, this is no longer widely employed owing to its lack of accuracy, sensitivity, and with regard to animal welfare. Therefore, alternative toxicity analyses have been developed based on MCs’ and OAs’ specific inhibition of protein phosphatase 2A (PP2A), using p-nitrophenylphosphate (p-NPP) as a substrate. The assay is simple, inexpensive, ready for use on site, and can be applied to several samples at once. For OA detection, this assay method is appropriate for widespread application as a substitute for MBA, as evidenced by its alignment with the oral toxicity of MBA. In this review, we summarize the structure and function of PP2A, the inhibitory activities of OAs and MCs against PP2A, and the practical applications of the PP2A assay, with the aim of improving understanding of the PP2A assay as an OAs and MCs detection and quantification method, as well as its suitability for screening before confirmatory chemical analysis. Full article

The Indian River Lagoon (IRL), a 156-mile-long estuary located on the eastern coast of Florida, experiences phytoplankton bloom events due to increased seasonal temperatures coupled with anthropogenic impacts. This study aimed to gather data on the toxicity to human cells and to identify secondary metabolites found in water samples collected in the IRL. Water samples from 20 sites of the IRL were collected during the wet and dry seasons over a three-year period. A panel of cell lines was used to test cytotoxicity. Hemagglutination, hemolysis, and inhibition of protein phosphatase 2A (PP2A) were also measured. Cytotoxic blooms were seen both in the south (Microcystis) and the north (Pyrodinium) of the IRL. Each toxin induced a consistent pattern of cytotoxicity in the panel of human cell lines assayed. During blooms, cytotoxicity due to a single type of toxin is obvious from this pattern. In the absence of blooms, the cytotoxicity seen reflected either a mixture of toxins or it was caused by an unidentified toxin. These observations suggest that other toxins with the potential to be harmful to human health may be present in the IRL. Moreover, the presence of toxins in the IRL is not always associated with blooms of known toxin-producing organisms. Full article

Reversible phosphorylation of proteins is a ubiquitous regulatory mechanism in vivo that can respond to external changes, and plays an extremely important role in cell signal transduction. Protein phosphatase 2C is the largest protein phosphatase family in higher plants. Recently, it has been found that some clade A members can negatively regulate ABA signaling pathways. However, the functions of several subgroups of Arabidopsis PP2C other than clade A have not been reported, and whether other members of the PP2C family also participate in the regulation of ABA signaling pathways remains to be studied. In this study, based on the previous screening and identification work of PP2C involved in the ABA pathway, the clade F member PIA1 encoding a gene of the PP2C family, which was down-regulated after ABA treatment during the screening, was selected as the target. Overexpression of PIA1 significantly down-regulated the expression of ABA marker gene RD29A in Arabidopsis protoplasts, and ABA-responsive elements have been found in the cis-regulatory elements of PIA1 by promoter analysis. When compared to Col-0, transgenic plants overexpressing PIA1 were less sensitive to ABA, whereas pia1 showed the opposite trait in seed germination, root growth, and stomatal opening experiments. Under drought stress, SOD, POD, CAT, and APX activities of PIA1 overexpression lines were lower than Col-0 and pia1, while the content of H₂O₂ was higher, leading to its lowest survival rate in test plants, which were consistent with the significant inhibition of the expression of ABA-dependent stress-responsive genes RD29B, ABI5, ABF3, and ABF4 in the PIA1 transgenic background after ABA treatment. Using yeast two-hybrid and luciferase complementation assays, PIA1 was found to interact with multiple ABA key signaling elements, including 2 RCARs and 6 SnRK2s. Our results indicate that PIA1 may reduce plant drought tolerance by functioning as a common negative regulator involved in ABA signaling pathway. Full article

Glucocorticoids inhibit angiogenesis in the femoral head, which fails to nourish the bone tissue and leads to osteonecrosis. Restoring angiogenesis is not only essential for vessel formation, but also crucial for osteogenesis. Poly (_L-lactic acid) (PLLA) is commonly used in the bone tissue engineering field. Panax notoginseng saponins (PNS) and osteopractic total flavone (OTF) promote angiogenesis and osteogenesis, respectively. We designed a sequentially releasing PLLA scaffold including PLLA loaded with OTF (inner layer) and PLLA loaded with PNS (outer layer). We assessed the osteogenic effect of angiogenesis in this scaffold by comparing it with the one-layered scaffold (PLLA embedded with OTF and PNS) in vivo. Results from the micro-CT showed that the data of bone mineral density (BMD), bone volume (BV), and percent bone volume (BV/TV) in the PO-PP group were significantly higher than those in the POP group (p < 0.01). Histological analyses show that the PO-PP scaffold exhibits better angiogenic and osteogenic effects compared with the one-layered scaffold. These might result from the different structures between them, where the sequential release of a bi-layer scaffold achieves the osteogenic effect of vascularization by initially releasing PNS in the outer layer. We further explored the possible mechanism by an immunohistochemistry analysis and an immunofluorescence assay. The results showed that the protein expressions of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1(CD31) in the PO-PP scaffold were significantly higher than those in the POP scaffold (p < 0.01); the protein expressions of osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) in the PO-PP scaffold were significantly higher than those in the POP scaffold (p < 0.05). Upregulating the expressions of angiogenic and osteogenic proteins might be the possible mechanism. Full article

Verticillium wilt, a soilborne disease caused by Verticillium dahliae (V. dahliae), can severely affect the yields of Solanaceae crops. In a previous study, it was observed in Solanum torvum (S. torvum) that protein phosphatase 5 (PP5) was induced by V. dahliae infection. To elucidate the function of PP5 more clearly, this study cloned an StPP5 cDNA from S. torvum by PCR. The cDNA contained an ORF of 1458 bp long encoding a putative protein of 485 amino acid residues with a predicted molecular mass of 54.63 kDa and a theoretical isoelectric point of 5.66. StPP5 protein contained a conserved PP domain and showed high similarity to other homologous members of the PP5 family from various plant species. The expression of StPP5 gene was upregulated after V. infection and reached its maximum value at 24 h in leaves. In order to clarify the role of StPP5, four transgenic tobacco plants expressing StPP5 were generated through Agrobacterium-mediated transformation and identified by PCR. In vitro culture assay showed that the growth of V. dahliae in PDA medium containing proteins extracted from the leaves of transgenic tobacco line P6 was inhibited, whose inhibition rate was 55.1%, higher than the non-transgenic control. These results indicated that StPP5 might be involved in plant defense against V. dahliae infection. Full article

Bud endodormancy is an important, complex process subject to both genetic and epigenetic control, the mechanism of which is still unclear. The endogenous hormone abscisic acid (ABA) and its signaling pathway play important roles in the endodormancy process, in which the type 2C protein phosphatases (PP2Cs) is key to the ABA signal pathway. Due to its excellent effect on endodormancy release, hydrogen cyanamide (HC) treatment is considered an effective measure to study the mechanism of endodormancy release. In this study, RNA-Seq analysis was conducted on endodormant floral buds of pear (Pyrus pyrifolia) with HC treatment, and the HC-induced PP2C gene PpPP2C1 was identified. Next, software prediction, expression tests and transient assays revealed that lncRNA PpL-T31511-derived Pp-miRn182 targets PpPP2C1. The expression analysis showed that HC treatment upregulated the expression of PpPP2C1 and downregulated the expression of PpL-T31511 and Pp-miRn182. Moreover, HC treatment inhibited the accumulation of ABA signaling pathway-related genes and hydrogen peroxide (H₂O₂). Furthermore, overexpression of Pp-miRn182 reduced the inhibitory effect of PpPP2C1 on the H₂O₂ content. In summary, our study suggests that downregulation of PpL-T31511-derived Pp-miRn182 promotes HC-induced endodormancy release in pear plants through the PP2C-H₂O₂ pathway. Full article

Environmental stimuli attack the skin daily resulting in the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein Phosphatase 2A (PP2A), a phosphatase which has been previously linked to Alzheimer’s Disease and aging. Oxidative stress decreases PP2A methylation in normal human dermal fibroblasts (NHDFs). Thus, we hypothesize agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a library of 32 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin, to be the most potent PP2A demethylating extract. Via several fractionation and extraction steps we developed a novel grape seed extract called Activated Grape Seed Extract (AGSE), which is enriched for PP2A activating flavonoids that increase potency in preventing PP2A demethylation when compared to commercial GSE. We then determined that 1% AGSE and 1% commercial GSE exhibit distinct gene expression profiles when topically applied to a 3D human skin model. To begin to characterize AGSE’s activity, we investigated its antioxidant potential and demonstrate it reduces ROS levels in NHDFs and cell-free assays equal to or better than Vitamin C and E. Moreover, AGSE shows anti-inflammatory properties, dose-dependently inhibiting UVA, UVB and chemical-induced inflammation. These results demonstrate AGSE is a novel, multi-functional extract that modulates methylation levels of PP2A and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin. Full article

Diarrhetic shellfish poisoning (DSP) is a globally occurring disease threatening public health and trade. The causative toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are collectively called OAs, and are quantified using the LC-MS/MS method. The hazardous effect of total OAs is expressed as the sum of OA equivalents defined for respective OAs based on mouse lethality, produced by either intraperitoneal (OAip) or oral administration (OAor). OAs are potent inhibitors of protein phosphatase 2A (PP2A) and are cytotoxic, necessitating expansion of the concept of OA equivalents to all relevant bioactivities. In this study, we determined OA equivalents for respective OA members in PP2A inhibition and cytotoxicity assays. To secure result credibility, we used certified OAs, reference materials, and PP2A produced using genetic engineering. The relative ratio of the OA equivalents determined by PP2A inhibition assays for OA, DTX1, and DTX2 were 1.0:1.6:0.3, while the ratio determined using the cytotoxicity assays indicated 1.0:1.5:0.5. OA equivalents showed a similar tendency in the PP2A inhibition and cytotoxicity assays, and matched better with oral toxicity data than intraperitoneal toxicity in mice. The PP2A inhibition assay, which measures the core activity of the OAs, suggested a higher OA equivalent for DTX1 than that currently used. Full article

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r² = 0.86) and the PP2A kit (r² = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time. Full article

The American cockroach, Periplaneta americana (L.), is a notorious urban pest. It has developed insecticidal resistance to commonly used insecticides. Cantharidin (CTD) is a defensive toxin derived from blister beetles. It has been verified to have insecticidal toxicity in a range of pests. In this study, we determined the ingestion toxicity of CTD and norcantharidin (NCTD) to P. americana to test whether they had the potential to be effective against P. americana. Bioassays revealed that CTD produces toxicity against P. americana. The median lethal concentration (LC₅₀) value of CTD was 50.92 μg/mL, while NCTD displayed nearly no toxicity against P. americana. The inhibition assays of serine/threonine protein phosphatases (PSPs) in P. americana indicated that CTD and NCTD could inhibit PSPs. The value of the half maximal inhibitory concentration (IC₅₀) of CTD was 7.21 ± 0.94 μM, whereas that of NCTD was higher, at 31.65 ± 3.87 μM. Furthermore, the inhibition effect of CTD on the serine/threonine protein phosphatase type 5 of P. americana (PaPP5) was superior to that of NCTD. Specifically, the IC₅₀ of CTD reached 0.39 ± 0.04 μM, while the IC₅₀ of NCTD was 1.87 ± 0.23 μM. This study paves the way for insect-derived agents (CTD) to be applied toward controlling P. americana and contributes to the development of novel insecticides based on PP5 as a target. Full article

Background: Colorectal cancer (CRC) is a high incidence of malignant tumors that lacks highly effective and targeted drugs and thus it is in urgent need of finding new specific molecular targets. Methods and Results: In this study, by using WST-1 (Highly water-soluble tetrazolium salt-1) and colony formation assays, we found that C20orf27 (chromosome 20 open reading frame 27), a functionally unknown protein, enhanced the growth and proliferation of CRC cells. The nude mouse tumor formation experiments verified that C20orf27 promoted the growth of CRC. Signal pathway analysis identified the TGFβR-TAK1-NFĸB cascade as a mediator in C20orf27-induced CRC progression. Inhibition experiments using NFĸB inhibitors reversed this progression. Co-immunoprecipitation showed that C20orf27 promoted the activation of the TGFβR-TAK1-NFĸB pathway by interacting with PP1c (the catalytic subunit of type 1 phosphatase). Conclusions: Our results firstly characterized the functional role and molecular mechanism of C20orf27 in driving CRC growth and proliferation through the TGFβR-TAK1-NFĸB pathway, suggesting its potential as a novel CRC candidate therapeutic target and tumor marker. Full article

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography–mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan. Full article