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PPM1F has been shown to play diverse biological functions in the progression of multiple tumors. PPM1F controls the T788/T789 phosphorylation switch of ITGB1 and regulates integrin activity. However, the impacts of PPM1F and ITGB1 on ovarian cancer (OV) progression remain unclear. Whether there is such a regulatory relationship between PPM1F and ITGB1 in ovarian cancer has not been studied. Therefore, the purpose of this study is to elucidate the function and the mechanism of PPM1F in ovarian cancer. The expression level and the survival curve of PPM1F were analyzed by databases. Gain of function and loss of function were applied to explore the function of PPM1F in ovarian cancer. A tumor formation assay in nude mice showed that knockdown of PPM1F inhibited tumor formation. We tested the effect of PPM1F on ITGB1 dephosphorylation in ovarian cancer cells by co-immunoprecipitation and western blotting. Loss of function was applied to investigate the function of ITGB1 in ovarian cancer. ITGB1-mut overexpression promotes the progression of ovarian cancer. Rescue assays showed the promoting effect of ITGB1-wt on ovarian cancer is attenuated due to the dephosphorylation of ITGB1-wt by PPM1F. PPM1F and ITGB1 play an oncogene function in ovarian cancer. PPM1F regulates the phosphorylation of ITGB1, which affects the occurrence and development of ovarian cancer.

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BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.

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Investigating the reactivity of small nucleophilic scaffolds is a strategic approach for the design of new catalysts aiming at effective detoxification processes of organophosphorus compounds. The drug methimazole (MMZ) is an interesting candidate featuring two non-equivalent nucleophilic centers. Herein, phosphoryl transfer reactions mediated by MMZ were assessed by means of spectrophotometric kinetic studies, mass spectrometry (MS) analyses, and density functional theory (DFT) calculations using the multi-electrophilic compound O,O-diethyl 2,4-dinitrophenyl phosphate (DEDNPP). MMZ anion acts primarily as an S-nucleophile, exhibiting a nucleophilic activity comparable to that of certain oximes featuring alpha-effect. Selective nucleophilic aromatic substitution was observed, consistent with the DFT prediction of a low energy barrier. Overall, the results bring important advances regarding the mechanistic understanding of nucleophilic dephosphorylation reactions, which comprises a strategic tool for neutralizing toxic organophosphates, hence promoting chemical security.

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The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs, undergo phosphorylation by PDK1 during their synthesis and subsequently get activated by DAG and Calcium. On the other hand, other substrates, including members of the Akt/PKB, S6K, SGK, and RSK families, undergo phosphorylation and activation downstream of PI3-kinase signaling. This review presents two accepted molecular mechanisms that determine the precise and timely phosphorylation of different substrates by PDK1. The first mechanism involves the colocalization of PDK1 with Akt/PKB in the presence of PIP3. The second mechanism involves the regulated docking interaction between the hydrophobic motif (HM) of substrates and the PIF-pocket of PDK1. This interaction, in trans, is equivalent to the molecular mechanism that governs the activity of AGC kinases through their HMs intramolecularly. PDK1 has been instrumental in illustrating the bi-directional allosteric communication between the PIF-pocket and the ATP-binding site and the potential of the system for drug discovery. PDK1's interaction with substrates is not solely regulated by the substrates themselves. Recent research indicates that full-length PDK1 can adopt various conformations based on the positioning of the PH domain relative to the catalytic domain. These distinct conformations of full-length PDK1 can influence the interaction and phosphorylation of substrates. Finally, we critically discuss recent findings proposing that PIP3 can directly regulate the activity of PDK1, which contradicts extensive in vitro and in vivo studies conducted over the years.

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Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and ßIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.

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A green approach to produce a cellulose-derived biocatalyst containing hydroxamic acids targeted for the neutralization of toxic organophosphates is shown. The cellulose source, rice husk, is among the largest agricultural waste worldwide and can be strategically functionalized, broadening its sustainable application. Herein, rice husk was oxidized in different degrees, leading to carboxylic acid-based colloidal and solid samples. These were functionalized with hydroxamic acids via amide bonds and fully characterized. The hydroxamic acid derived biocatalysts were evaluated in the cleavage of toxic organophosphates, including the pesticide Paraoxon. Catalytic increments reached up to 107-fold compared to non-catalyzed reactions. Most impressively, the materials showed P atom-selectivity and recyclability features. This guarantees only one reaction pathway that leads to less toxic products, hereby, detoxifies. Overall, highly sustainable catalysts are presented, that benefits from waste source, its green functionalization and is successfully employed for the promotion of chemical security of threatening organophosphates. To the best of our knowledge, this is the first report of a hydroxamate-derived rice husk (selectively modified at the C6 of cellulose) and its application in organophosphates reaction.

Assuntos

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Neutralization of organophosphates is an issue of public health and safety, involving agrochemicals and chemical warfare. A promising approach is the nucleophilic neutralization, scope of this review, which focuses on the molecular nucleophiles: hydroxide, imidazole derivatives, alpha nucleophiles, amines and other nucleophiles. A reactivity mapping is given correlating the pathways and reaction efficiency with structural dependence of the nucleophile (basicity) and the organophosphate (electrophilic centers, P=O/P=S shift, leaving and non-leaving group). Reactions extremely unfavorable (>20 years) can be reduced to seconds with various nucleophiles, some which are catalytic. Although there is no universal nucleophile, a lack of selectivity in some cases accounts for plenty of versatility in other reactions. The ideal neutralization requires a solid mechanistic understanding, together with balancing factors such as milder conditions, fast process, selectivity and less toxic products.

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The dual-specificity phosphatase 3 (DUSP3), an atypical protein tyrosine phosphatase (PTP), regulates cell cycle checkpoints and DNA repair pathways under conditions of genotoxic stress. DUSP3 interacts with the nucleophosmin protein (NPM) in the cell nucleus after UV-radiation, implying a potential role for this interaction in mechanisms of genomic stability. Here, we show a high-affinity binding between DUSP3-NPM and NPM tyrosine phosphorylation after UV stress, which is increased in DUSP3 knockdown cells. Specific antibodies designed to the four phosphorylated NPM's tyrosines revealed that DUSP3 dephosphorylates Y29, Y67, and Y271 after UV-radiation. DUSP3 knockdown causes early nucleolus exit of NPM and ARF proteins allowing them to disrupt the HDM2-p53 interaction in the nucleoplasm after UV-stress. The anticipated p53 release from proteasome degradation increased p53-Ser15 phosphorylation, prolonged p53 half-life, and enhanced p53 transcriptional activity. The regular dephosphorylation of NPM's tyrosines by DUSP3 balances the p53 functioning and favors the repair of UV-promoted DNA lesions needed for the maintenance of genomic stability.

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The harmful impact caused by pesticides on human health and the environment necessitates the development of efficient degradation processes and control of prohibited stocks of such substances. Organophosphates (OPs) are among the most used agrochemicals in the world and their degradation can proceed through several possible pathways. Investigating the reactivity of OPs with nucleophilic species allows one to propose new and efficient catalyst scaffolds for use in detoxification. In light of the remarkable catalytic activity of imidazole (IMZ) at promoting dephosphorylation processes of OPs, the reactivity of 4(5)-hydroxymethylimidazole (HMZ) with diethyl-2,4-dinitrophenylphosphate (DEDNPP) and Paraoxon are evaluated by combining experimental and theoretical approaches. It is observed that HMZ is an efficient and regiospecific catalyst with reactivity modulated by competing tautomers. To propose an optimal IMZ-based catalyst, quantum chemical calculations were performed for monosubstituted 4(5)IMZ derivatives that might cleave DEDNPP. Both inductive effects and hydrogen bonding by the substituents are shown to influence barriers and mechanisms.

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The distributive sequential n-site phosphorylation/dephosphorylation system is an important building block in networks of chemical reactions arising in molecular biology, which has been intensively studied. In the nice paper of Wang and Sontag (2008) it is shown that for certain choices of the reaction rate constants and total conservation constants, the system can have 2[n/2] +1 positive steady states (that is, n+1 positive steady states for n even and n positive steady states for n odd). In this paper we give open parameter regions in the space of reaction rate constants and total conservation constants that ensure these number of positive steady states, while assuming in the modeling that roughly only 1/4 of the intermediates occur in the reaction mechanism. This result is based on the general framework developed by Bihan, Dickenstein, and Giaroli (2018), which can be applied to other networks. We also describe how to implement these tools to search for multistationarity regions in a computer algebra system and present some computer aided results.

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Aiming to clarify the mechanism of inhibition of (Na+, K+)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na+, K+)-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s-1) in adults than in juveniles (0.053 ± 0.003 s-1) for spermidine, but similar to juveniles (0.059 ± 0.004 s-1) for putrescine. Maximum phosphointermediate formation for the (Na+, K+)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na+, K+)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

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An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 (5) s (-1) and 4.7 × 10 (6) s (-1) .M (-1) , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg (2+) , Cd (2+) , K (+) and Ca (2+) , and it was drastically inhibited by F (-) . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 µmol phosphate/mL after 2.5 h of treatment.

Assuntos

RESUMO

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

Assuntos

RESUMO

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.(AU)

Assuntos

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AIM: The Na(+) /H(+) exchanger NHE3 activity decreases in the proximal tubule of spontaneously hypertensive rats (SHRs) as blood pressure increases, and this reduction is correlated with higher NHE3 phosphorylation levels at the PKA consensus site serine 552. This study tested the hypothesis that this lowered NHE3 activity is associated with an increase in PKA activity and expression, and/or a decrease in protein phosphatase-1 (PP1) activity and expression. METHODS: Proximal tubule NHE3 activity was measured as the rate of bicarbonate reabsorption by stationary microperfusion. NHE3 phosphorylation and protein expression were determined by immunoblotting. PKA and PP1 activities were determined using specific substrates under optimal enzymatic conditions. RESULTS: The PKA activator, 6-MB-cAMP, increased the phosphorylation levels of NHE3 at serine 552 in the renal cortex; this increase happens to a much greater extent in young pre-hypertensive SHRs (Y-SHRs) compared to adult SHRs with established hypertension (A-SHRs). Likewise, the inhibitory effect of 6-MB-cAMP on NHE3 transport activity was much more pronounced in the proximal tubules of Y-SHRs than in those of A-SHRs. Renal cortical activity of PKA was not significantly different between Y-SHRs and A-SHRs. On the other hand, Y-SHRs exhibited higher protein phosphatase 1 (PP1) activity, and their expression of the PP1 catalytic subunit PP1α in the renal cortex was also higher than in A-SHRs. CONCLUSION: Collectively, these results support the idea that the lower NHE3 transport activity and higher phosphorylation occurring after the development of hypertension in SHRs are due, at least in part, to reduced PP1-mediated dephosphorylation of NHE3 at serine 552.

Assuntos

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The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.

RESUMO

The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.

RESUMO

The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.