PTP1B Activity Research Articles

Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction.

Protein-protein interactions are critical for cell signaling. The interaction between the phosphatase PTP1B and adaptor protein Grb2 co-localizes PTP1B with its substrates, thereby enhancing their dephosphorylation. We show that Grb2 binding also directly modulates PTP1B activity through an allosteric mechanism involving the proline-rich region of PTP1B. Our study reveals a novel mode of PTP1B regulation through a protein-protein interaction that is likely to be exploited by other cellular interactors of this important signaling enzyme.

Circulating extracellular vesicle-carried PTP1B and PP2A phosphatases as regulators of insulin resistance.

Metabolic disorders associated with abdominal obesity, dyslipidaemia, arterial hypertension and hyperglycaemia are risk factors for the development of insulin resistance. Extracellular vesicles (EVs) may play an important role in the regulation of metabolic signalling pathways in insulin resistance and associated complications. Circulating large EVs (lEVs) and small EVs (sEVs) from individuals with (IR group) and without insulin resistance (n-IR group) were isolated and characterised. lEVs and sEVs were administered by i.v. injection to mice and systemic, adipose tissue and liver insulin signalling were analysed. The role of phosphatases was analysed in target tissues and cells. Injection of lEVs and sEVs from IR participants impaired systemic, adipose tissue and liver insulin signalling in mice, while EVs from n-IR participants had no effect. Moreover, lEVs and sEVs from IR participants brought about a twofold increase in adipocyte size and adipogenic gene expression. EVs from IR participants expressed two types of phosphatases, phosphotyrosine 1 phosphatase (PTP1B) and protein phosphatase 2 (PP2A), IR lEVs being enriched with the active form of PTP1B while IR sEVs mainly carried active PP2A. Blockade of PTP1B activity in IR lEVs fully restored IRS1 and Akt phosphorylation in adipocytes and blunted insulin-induced Akt phosphorylation by inhibition of the macrophage secretome in hepatocytes. Conversely, blockade of PP2A activity in IR sEVs completely prevented insulin resistance in adipocytes and hepatocytes. These data demonstrate that inhibition of phosphatases carried by EVs from IR participants rescues insulin signalling in adipocytes and hepatocytes and point towards PTP1B and PP2A carried by IR EVs as being novel potential therapeutic targets against insulin resistance in adipose tissue and liver and the development of obesity.

Oleanane triterpenoids with PTP1B and α-glucosidase inhibitory activities from Caulophyllum robustum

Eleven oleanane triterpenoids (1–11) including two new ones (1 and 2) were isolated from the roots and stems of Caulophyllum robustum. Their structures were established by extensive spectroscopic analysis, comparison with literature, and NMR calculations. Compounds 1 and 2 represent the first examples of 23-hydroxy-28-nor-oleanane and 21-hydroxy-olean-3-one triterpenoids, respectively. All isolates were evaluated for their PTP1B and α-glucosidase inhibitory activities in vitro. Among them, the triterpene aglycones 1–5 showed almost equivalent PTP1B inhibitory activities to oleanolic acid and ursolic acid, while 1, 2, and the triterpene saponins 6–11 showed significant α-glucosidase inhibitory activities. Furthermore, compounds 1 and 3 were proved to regulate the expression of proteins implicated the PTP1B/IRS-1/pIRS-1 signalling pathway to improve insulin resistance.

Effect of Lampaya medicinalis Phil. (Verbenaceae) and Palmitic Acid on Insulin Signaling and Inflammatory Marker Expression in Human Adipocytes.

Aging and obesity are associated with insulin resistance (IR) and low-grade inflammation. Molecularly, IR is characterized by a reduction in glucose uptake and insulin signaling (IRS-1/Akt/AS160 pathway), while inflammation may result from upregulated NF-κB pathway after low Tyr-IκBα phosphorylation. Upregulated phosphatase activity of PTP1B is associated with impaired insulin signaling and increased inflammation. Plasma levels of palmitic acid (PA) are elevated in obesity, triggering inflammation and disruption of insulin signaling. Traditional medicine in Northern Chile uses oral infusions of Lampaya medicinalis Phil. (Verbenaceae) to treat inflammatory conditions. Significant amounts of flavonoids are found in the hydroethanolic extract of Lampaya (HEL), which may account for its biological activity. The aim of this work was to study the effect of HEL and PA on insulin signaling and glucose uptake as well as inflammatory marker expression in human adipocytes. We studied HEL effects on PA-induced impairment on insulin signaling, glucose uptake and inflammatory marker content in human SW872 adipocytes. HEL cytotoxicity was assessed in adipocytes at different concentrations (0.01 to 10 g/mL). Adipocytes were incubated or not with PA (0.4 mM, 24 h) with or without HEL (2 h pre-incubation), and then stimulated with insulin (10 min, 100 mM) or a vehicle. Phospho-IRS-1, phospho-Akt, phospho-AS160, phospho-NF-κB and phospho-IκBα, as well as protein levels of PTP1B, were assessed using Western blotting, and glucose uptake was evaluated using the 2-NBDG analogue. At the assessed HEL concentrations, no cytotoxic effects were observed. PA decreased insulin-stimulated phospho-Akt and glucose uptake, while co-treatment with HEL increased such markers. PA decreased phospho-IRS-1 and phospho-Tyr-IκBα. On the other hand, incubation with HEL+PA decreased phospho-AS160 and phospho-NF-κB compared with cells treated with PA alone. Our results suggest a beneficial effect of HEL by improving PA-induced impairment on molecular markers of insulin signaling, glucose uptake and inflammation in adipocytes. Further studies are necessary to elucidate whether lampaya may constitute a preventive strategy for people whose circulating PA levels contribute to IR and inflammation during aging and obesity.

Multiscale In Silico Study of the Mechanism of Activation of the RtcB Ligase by the PTP1B Phosphatase.

Inositol-requiring enzyme 1 (IRE1) is a transmembrane sensor that is part of a trio of sensors responsible for controlling the unfolded protein response within the endoplasmic reticulum (ER). Upon the accumulation of unfolded or misfolded proteins in the ER, IRE1 becomes activated and initiates the cleavage of a 26-nucleotide intron from human X-box-containing protein 1 (XBP1). The cleavage is mediated by the RtcB ligase enzyme, which splices together two exons, resulting in the formation of the spliced isoform XBP1s. The XBP1s isoform activates the transcription of genes involved in ER-associated degradation to maintain cellular homeostasis. The catalytic activity of RtcB is controlled by the phosphorylation and dephosphorylation of three tyrosine residues (Y306, Y316, and Y475), which are regulated by the ABL1 tyrosine kinase and PTP1B phosphatase, respectively. This study focuses on investigating the mechanism by which the PTP1B phosphatase activates the RtcB ligase using a range of advanced in silico methods. Protein-protein docking identified key interacting residues between RtcB and PTP1B. Notably, the phosphorylated Tyr306 formed hydrogen bonds and salt bridge interactions with the "gatekeeper" residues Arg47 and Lys120 of the inactive PTP1B. Classical molecular dynamics simulation emphasized the crucial role of Asp181 in the activation of PTP1B, driving the conformational change from an open to a closed state of the WPD-loop. Furthermore, QM/MM-MD simulations provided insights into the free energy landscape of the dephosphorylation reaction mechanism of RtcB, which is mediated by the PTP1B phosphatase.

Inhibitory Activity of PTP1B and α-Glucosidase by Compounds from Whole Plants of Houttuynia cordata Thunb.

Inhibitory Activity of PTP1B and α-Glucosidase by Compounds from Whole Plants of Houttuynia cordata Thunb.

Discovery and biological evaluation of novel dual PTP1B and ACP1 inhibitors for the treatment of insulin resistance

In this study, a virtual screening pipeline comprising ligand-based and structure-based approaches was established and applied for the identification of dual PTP1B and ACP1 inhibitors. As a result, a series of benzoic acid derivatives was discovered, and compound H3 and S6 demonstrated PTP1B and ACP1 inhibitory activity, with IC50 values of 3.5 and 8.2 μM for PTP1B, and 2.5 and 5.2 μM for ACP1, respectively. Molecular dynamics simulations illustrated that H3 interacted with critical residues in the active site, such as Cys215 and Arg221 for PTP1B, and Cys17 and Arg18 for ACP1. Enzymatic kinetic research indicated that identified inhibitors competitively inhibited PTP1B and ACP1. Additionally, cellular assays demonstrated that H3 and S6 effectively increased glucose uptake in insulin-resistant HepG2 cells while displaying very limited cytotoxicity at their effective concentrations. In summary, H3 and S6 represent novel dual-target inhibitors for PTP1B and ACP1, warranting further investigation as potential agents for the treatment of diabetes.

Pharmacological properties of Ensete glaucum seed extract: Novel insights for antidiabetic effects via modulation of oxidative stress, inflammation, apoptosis and MAPK signaling pathways

Ethnophamacological relevanceMedicinal plants are increasingly making important contributions to diabetic treatment. Ensete glaucum seeds have been widely used in folk medicine to treat diabetes. Aim of the studyThe study was aimed to investigate the protective effect and active mechanisms of E. glaucum seed extract (EGSE) against streptozotocin (STZ)-induced hyperglycemia. Materials and methodsHyperglycemic mice were treated with EGSE (25 and 50 mg/kg) or glibenclamide (5 mg/kg) once daily for 7 d. The effects of these treatments on changes in blood biochemical parameters, pancreatic, liver, and kidney histopathology, oxidative stress and inflammatory marker levels in pancreatic, hepatic, and renal tissues were assessed. Expression of several proteins in MAPK signaling pathway related to apoptosis in pancreatic tissue were investigated. Furthermore, ex vivo, in vitro, and in silico biological activities of EGSE and its compounds were also examined. ResultsEGSE and glibenclamide increased notably insulin, reduced significantly glucose, AST, ALT, BUN and creatinine levels in blood. Pancreatic islets, hepatic and renal tissue structure were restored by EGSE or glibenclamide. EGSE showed significant anti-oxidative stress and anti-inflammatory effects by enhancing GSH level and dropping MDA, NF-κB, TNF-α and IL-6 levels in these tissues. Particularly, EGSE exhibited pancreatic protective effect against STZ-induced apoptosis through the MAPK signaling pathway by down-regulation of p-p38 MAPK, ERK1/2, JNK1, p-AMPK, Bax, Bax/Bcl-2, cytochrome c, cleaved-caspase 3 and PARP expression, and slight up-regulation of Bcl-2 expression. Moreover, EGSE inhibited intestinal glucose absorption, PTP1B, α-amylase, and α-glucosidase activities. Its isolated compounds (Afzelechin and coniferaldehyde) showed PTP1B and α-glucosidase inhibitory activities, and potent structure-activity relationships. ConclusionThese findings indicated the hypoglycemic and protective effects of E. glaucum seed extract against the STZ diabetogenic action. E. glaucum seed is a potential candidate for further studies to confirm its activities as a therapeutic agent for diabetic patients.

Myeloid PTP1B deficiency protects against atherosclerosis by improving cholesterol homeostasis through an AMPK-dependent mechanism

ObjectiveAtherosclerosis is a chronic inflammatory process induced by the influx and entrapment of excess lipoproteins into the intima media of arteries. Previously, our lab demonstrated that systemic PTP1B inhibition protects against atherosclerosis in preclinical LDLR−/− models. Similarly, it was shown that myeloid-specific PTP1B ablation decreases plaque formation and ameliorates dyslipidaemia in the ApoE−/− model of atherosclerosis. We hypothesized that the relevant improvements in dyslipidaemia following modification of PTP1B activation may either result from changes in hepatic cholesterol biosynthesis and/or increased uptake and degradation by liver-resident macrophages. We examined this in animal models and patients with coronary artery disease.MethodsIn this study, we determined the cholesterol-lowering effect of myeloid-PTP1B deletion in mice fed a high-fat high-cholesterol diet and examined effects on total cholesterol levels and lipoprotein profiles. We also determined the effects of PTP1B inhibition to oxLDL-C challenge on foam cell formation and cholesterol efflux in human monocytes/macrophages.ResultsWe present evidence that myeloid-PTP1B deficiency significantly increases the affinity of Kupffer cells for ApoB containing lipoproteins, in an IL10-dependent manner. We also demonstrate that PTP1B inhibitor, MSI-1436, treatment decreased foam cell formation in Thp1-derived macrophages and increased macrophage cholesterol efflux to HDL in an AMPK-dependent manner. We present evidence of three novel and distinct mechanisms regulated by PTP1B: an increase in cholesterol efflux from foam cells, decreased uptake of lipoproteins into intra-lesion macrophages in vitro and a decrease of circulating LDL-C and VLDL-C in vivo.ConclusionsOverall, these results suggest that myeloid-PTP1B inhibition has atheroprotective effects through improved cholesterol handling in atherosclerotic lesions, as well as increased reverse cholesterol transport.Trial registration Research registry, researchregistry 3235. Registered 07 November 2017, https://www.researchregistry.com/browse-the-registry#home/registrationdetails/5a01d0fce7e1904e93e0aac5/.

Diterpenoid and phenolic constituents from corn silk (Zea mays) with PTP1B inhibitory activity

Chemical investigation of corn silk resulted in the isolation of nine secondary metabolites, including a new ent-kaurane diterpenoid, zeamaysditerpene A (1) and eight known compounds, stigmaydene A (2), stigmaydene J (3), stigmaydene L (4), stigmane D (5), demethyltorosaflavone D (6), chrysoeriol 6-C-β-boivinopyranosyl-7-O-β-D-glucopyranoside (7), deoxypodophyllotoxin (8), and α-peltatin glucoside (9). Their structures were elucidated using a combination of spectroscopic methods, including 1D and 2D NMR and HRESIQTOF mass spectra. The absolute configuration of 1 was deduced by applying electronic circular dichroism (ECD) calculation method. Among the isolates, only 6 displayed significant inhibition against PTP1B activity in a dose-dependent manner, with an IC50 value of 10.7 ± 0.1 µM. Furthermore, molecular docking simulation was carried out to explore the action perspective of 6 inside the enzyme PTP1B. This finding suggests that 6 might be a potential lead for the development of a new anti-diabetic agent.

Carbon dots with aggregation-induced emission enhancement (AIEE) for detection of Zr4+/ Hf4+ and PTP1B activity

The frequent use of Zirconium (Zr) and Hafnium (Hf) in modern industries may result in serious environment issues, and thus developing analytical methods to facilitate the control of these two resembled metal elements is urgently needed. However, up to now, rapidly and conveniently detecting Zr4+ and Hf4+ is still full of challenge. In this study, nitrogen and sulfur co-doped carbon dots (N, S-Cdots) with aggregation-induced emission enhancement (AIEE) were prepared and used for turn-on detection of Zr4+ and Hf4+ within 10 min. The photoluminescent intensity of N, S-Cdots showed a good linear correlation with Zr4+ and Hf4+concentrations ranging from 0 to 30 μM with the limit of detection (LOD) of 0.47 and 0.53 μM, respectively. Furthermore, this fluorometric assay was successfully used for quantitative analysis of Zr4+ and Hf4+ in real water samples with satisfactory recoveries in the range of 91.16–108.50% and 90.28–106.49%, respectively. Finally, the developed N, S-Cdots sensing system was used for assay PTP1B activity and screening its inhibitor with Zr4+ as the medium. Our work demonstrated that the as-prepared N, S-Cdots with AIEE can offer a simple and reliable alternative for rapid detection of Zr4+ and Hf4+ in water samples, in addition to being potential useful in phosphatase analysis and drug discovery.

Oral sub-chronic treatment with Terminalia phaeocarpa Eichler (Combretaceae) reduces liver PTP1B activity in a murine model of diabetes.

The endemic Brazilian medicinal plants of the genus Terminalia (Combretaceae), popularly known as capitão, comprising the similar species Terminalia phaeocarpa Eichler and Terminalia argentea, are traditionally and indistinguishably used in the country to treat diabetes. The present work investigated the effect of 28 days of treatment with the crude ethanolic extract (CEE) and its derived ethyl acetate fraction (EAF) from T. phaeocarpa leaves in a mice model of diabetes. Streptozotocin-nicotinamide-fructose diabetic model was used to evaluate the antidiabetic activity of 28 days of treatment with the CEE and EAF from the leaves T. phaeocarpa and metformin as a positive control. Serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were measured with specific commercial kits and glucose with a strip glucometer. The thiobarbituric acid method measured the liver MDA level, while a colorimetric assay measured the GSH level and PTP1B activity. A UPLC-DAD profile was obtained to identify the main polyphenolic compound in the EAF. Treatment with CEE and EAF reduced plasma glucose in diabetic mice. At the end of the treatment, the plasma glucose level was significantly lower in EAF-treated (100 mg/kg) diabetic mice (106.1 ± 13.7 mg/dL) than those treated with 100 mg/kg CEE (175.2 ± 20.9 mg/dL), both significantly lower than untreated diabetic mice (350.4 ± 28.1 mg/dL). The serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were significantly reduced in diabetic mice treated with CEE and EAF. In the livers of diabetic mice, the treatment with CEE and EAF reduced MDA levels and the activity of the enzyme PTP1B (96.91 ± 3.7%, 113.8 ± 2.8%, and 134.8 ± 4.6% for CEE-, EAF-treated, and untreated diabetic mice, respectively). Galloylpunicalagin was the main polyphenol observed in the EAF of T. phaeocarpa. The present results demonstrate the significant antidiabetic effect of CEE and EAF of T. phaeocarpa and their reduction on the markers of liver dysfunction in diabetic mice. Moreover, the antidiabetic activity of T. phaeocarpa might be associated with lowering the augmented activity of the PTP1B enzyme in the liver of diabetic mice.

Effects of dietary wild bitter melon (Momordica charantia var. abbreviate Ser.) extract on glucose and lipid metabolism in HFD/STZ-induced type 2 diabetic rats.

Plant-based extracts to interfere with the onset of diabetes may be a promising approach towards type 2 diabetes mellitus (T2DM). Bitter gourd (Momordica charantia L.) is popularly consumed as an edible and medicinal resource with hypoglycemic effect in China. Wild bitter gourd (Momordica Charantia var. abbreviata Ser.) is a variant of bitter gourd, but there are relatively few studies on it. The purpose of the experiment is to first screen out the most effective extraction part of Momordica charantia L. and Momordica Charantia var. abbreviata Ser. through the hypoglycemic activity experiment in vitro, and by using a high-fat and high-sugar diet with STZ-induced diabetic rat model in vivo to explore the possible mechanism of action against diabetes. This study first performed α-glucosidase, PTP1B and lipase activities inhibition experiments on the alcohol and water extracts of Momordica charantia L. and Momordica Charantia var. abbreviata Ser. Sprague Dawley rats were either given normal feed or a high sugar and fat diet for four weeks, followed STZ (25 mg/kg, via i. p.) was given. Rats with fasting blood glucose ≥11.1 mmol/l after one week were deemed to be diabetic, treatments were administered for four weeks, and then blood samples were used to evaluate hematological and biochemical indicators, and liver was removed for post-analysis. The expression levels of p-AMPK, AMPK, p-PI3K, PI3K, p-AKT, AKT, p-GSK3β, GSK3β, p-IRS-1, IRS-1, GLUT2 were determined by Western blot. At the same time, the chemical components was identified by liquid-mass spectrometry. Data showed that the ethanol extract of wild bitter gourd (WBGE) had the best ability to regulate glucose and lipid metabolism in vitro. Therefore, we further investigated the antidiabetic effects of oral consumption of WBGE on high-fat diet (HFD) and streptozotocin (STZ)-induced T2DM in SD rats. WBGE effectively reduced blood glucose and lipid levels, alleviated glucose intolerance and insulin resistant. Moreover, WBGE consumption could also inhibited oxidant responses and inflammatory damage. Mechanism studies have shown that WBGE may act by regulating AMPK/PI3K signaling pathway. On the other hand, the content of total phenol, total flavonoids, total saponins and total polysaccharide were measured by UV, 27 compounds were identified by LC-MS. These studies explored the role and mechanism of WBGE in regulating glucose and lipid metabolism, and may support the utilization and further investigation of wild bitter gourd as a dietary intervention strategy to prevent diabetes and related metabolic abnormalities.

H2S- and Redox-State-Mediated PTP1B S-Sulfhydration in Insulin Signaling.

Because hydrogen sulfide (H2S) is classified as a gaseous signaling molecule, protein S-sulfhydration is known to be one of the mechanisms by which H2S signals are conducted. PTP1B, a negative regulator in insulin signaling, has been found to be S-sulfhydrated at Cys215-SH to form Cys215-SSH in response to endoplasmic reticulum (ER) stress. Therefore, we aimed to understand the change in PTP1B S-sulfhydration and cellular redox homeostasis in response to insulin stimulation. We demonstrated a feasible PEG-switch method to determine the levels of PTP1B S-sulfhydration. According to the results obtained from HEK293T and MDA-MB-231 cells, insulin induced a change in PTP1B S-sulfhydration that was similar to the change in Insulin receptor substrate 1 (IRS1) phosphorylation in both cell lines. However, insulin-induced PTP1B S-sulfhydration and IRS1 phosphorylation were only significantly affected by metformin in HEK293T cells. Insulin also induced an increase in reactive oxygen species (ROS) in both cell lines. However, the level of H2S, GSH, and GSSG was only significantly affected by insulin and metformin in HEK293T cells. HEK293T cells maintained high levels of H2S and cysteine, but low levels of GSSG and GSH in general compared to MDA-MB-231 cells. From these findings, we suggest that PTP1B activity is modulated by H2S and redox-regulated S-sulfhydration during insulin signaling.

Molecular targets of endothelial phosphatidic acid regulating major depressive disorder.

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis with a lifetime prevalence of ~10%. Therapy requires prolonged treatment that often fails. We have previously demonstrated that ceramide levels in the blood plasma of patients and in mice with experimental MDD are increased. Neutralization of blood plasma ceramide prevented experimental MDD in mice. Mechanistically, we demonstrated that blood plasma ceramide accumulated in endothelial cells of the hippocampus, inhibited phospholipase D (PLD) and thereby decreased phosphatidic acid in the hippocampus. Here, we demonstrate that phosphatidic acid binds to and controls the activity of phosphotyrosine phosphatase (PTP1B) in the hippocampus and thus determines tyrosine phosphorylation of a variety of cellular proteins including TrkB. Injection of PLD, phosphatidic acid, or inhibition of PTP1B abrogated MDD and normalized cellular tyrosine phosphorylation, including phosphorylation of TrkB and neurogenesis in the hippocampus. Most importantly, these treatments also rapidly normalized behavior of mice with experimental MDD. Since phosphatidic acid binds to and inhibits PTP1B, the lack of phosphatidic acid results in increased activity of PTP1B and thereby in reduced tyrosine phosphorylation of TrkB and other cellular proteins. Thus, our data indicate a novel pathogenetic mechanism of and a rapidly acting targeted treatment for MDD.

Betulinic acid improves TNF-α-induced insulin resistance by inhibiting negative regulator of insulin signalling and inflammation-activated protein kinase in 3T3-L1 adipocytes

Context Obesity is related to insulin resistance, and adipose tissue-secreted TNF-α may play a role in inducing obesity. TNF-α activates inflammatory protein kinase and impairs insulin signalling. Objectives We investigated the effect of betulinic acid on insulin resistance caused by TNF-α treatment in 3T3-L1 adipocytes. Material and methods 3T3-L1 was exposed to TNF-α in the presence and absence of betulinic acid. Various parameters such as glucose uptake assay, cell viability, expression of proteins involved in insulin resistance were studied. Results Betulinic acid increased glucose uptake in TNF-α pre-treated cells and inhibited the activation of PTP1B and JNK and reduced IκBα degradation. Tyrosine phosphorylation was increased, and serine phosphorylation was decreased in IRS-1. Discussion Betulinic acid restored TNF-α impaired insulin signalling and increased PI3K activation and phosphorylation of Akt and increased plasma membrane expression of GLUT 4, which stimulated glucose uptake concentration-dependently. Conclusion These results suggest that betulinic acid is effective at improving TNF-α-induced insulin resistance in adipocytes via inhibiting the activation of negative regulator of insulin signalling and inflammation-activated protein kinase and may potentially improve insulin resistance.

Argonaute 2 modulates EGFR-RAS signaling to promote mutant HRAS and NRAS-driven malignancies.

Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS-driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS/NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS/NRAS-driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by β-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR–MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR–AGO2–RAS signaling axis is essential for maintaining mutant HRAS and NRAS-driven malignancies.

Meroterpenoids with inhibitory activity of PTP1B from the fruits of Psidium guajava

Four new meroterpenoids (1–4) together with 11 known meroterpenoids (5–15) were isolated from the fruits of Psidium guajava Linn.. Their structures were elucidated by extensive spectroscopic data analysis, including HR-ESIMS, 1D and 2D-NMR, IR, X-ray and ECD data. In the bioactivity assay, compounds 2, 5, 11, and 12 exhibited significant inhibitory activities of PTP1B with IC50 values of 4.46, 8.67, 3.19 and 4.49 μM, respectively. The above results showed that the meroterpenoids from the fruits of guava had inhibitory activity of PTP1B and could be used for the development of anti-diabetic agents.

Lateral flow immunoassay with peptide-functionalized gold nanoparticles for rapid detection of protein tyrosine phosphatase 1B

In this work, a lateral flow immunoassay (LFIA) with peptide functionalized gold nanoparticles (termed as biotin-ppeptide-AuNPs) has been developed for rapid, semi-quantitative detection of PTP1B activity without using any sophisticated equipment. In this method, the anti-phosphotyrosine (anti-pY) monoclonal antibody and streptavidin were used as test line and control line, respectively. The biotin-ppeptide-AuNPs contain 10% biotinylated peptide ligand carry a motif SDGHEpYIYVDP with pY (phosphotyrosine) and 90% pentapeptide (CALNN) ligand, which are used as PTP1B substrates and LFIA labelling probes. The experimental results demonstrate that the as-proposed LFIA with biotin-ppeptide-AuNPs exhibits a wide linear range (from 50 ng/mL to 10 μg/mL), a relatively low limit of detection (LOD, 44 ng/mL), and good specificity. In addition, the LFIA with biotin-ppeptide-AuNPs has been successfully used to evaluate activity levels of PTP1B in four cell lysates and the detection results exhibit a consistent trend with that of commercial kit.

Regulation of Nrf2 and NF-κB activities may contribute to the anti-inflammatory mechanism of xylopic acid.

Xylopic acid (XA) is a kaurene diterpene which naturally exists in African plants such as Xylopia aethiopica. It has been established to exhibit acute and chronic anti-inflammatory activities from our earlier studies. This current work sets out to shed light on the potential molecular target(s) of xylopic acid. Selection of investigated targets (NF-κB, Nrf2 and PTP1B) was based on an unbiased approach, using the SPiDER in silico prediction tool, and a candidate approach, examining well-known anti-inflammatory targets. Reporter gene assays were used to test for altered NF-κB and Nrf2 activities in transfected HEK or CHO cells, respectively, and immunoblot and flow cytometric analyses examined protein expression of the Nrf2/NF-kB target genes HO-1 and VCAM-1 in HUVEC. An effect of XA on PTP1B activity assay was studied using an in vitro enzyme assay with recombinant human enzyme and pNPP as substrate as well as by looking at insulin receptor phosphorylation in HepG2 cells. XA at 30µM significantly (p < 0.001) inhibited the NF-κB-dependent reporter gene expression and enhanced activation of Nrf2 in a concentration-dependent manner when compared to the control. XA also marginally increased HO-1 protein expression levels while expression of VCAM-1 was reduced to 70% in XA-treated endothelial cells. However, XA did not show any sign of inhibition of PTP1B or a related phosphatase. Our findings suggest that the anti-inflammatory mechanism of XA entails the inhibitory effect on NF-κB and an increased activity of Nrf2, accompanied by increased expression of HO-1 and reduced expression of VCAM-1.