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Enzymatic activity and inhibition, thermal stability and electrophoretic properties of induced and constitutive acid phosphatases of Euglena gracilis
Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.
Composition and diversity of vegetation systems can influence soil microbial activity and extracellular enzyme (EE) dynamics, which are crucial for soil carbon (C) accrual and nutrient cycling. Yet, the impact of plant interactions and competition on EE activities remains a notable knowledge gap. This study examines how direct root contact and neighboring plant identity affect the activity and spatial distribution of four key soil EEs: β-glucosidase (BGlu), chitinase, acid phosphatase (AcidP), and alkaline phosphatase (AlkP). Using three-compartment rhizoboxes with switchgrass (Panicum virgatum L.) grown alongside bush clover (Lespedeza capitata Michx.), and black-eyed Susan (Rudbeckia hirta L.), we assessed enzyme activities using zymography under conditions that either allowed or restricted direct root contact by root barriers. Results show that root proliferation and species interactions significantly influenced EE activity. While BGlu and AcidP activities were strongly correlated with root biomass, AlkP activity was consistently higher in the absence of root barriers, indicating a pronounced microbial response to plant interactions via direct/close root contacts. Additionally, soil phosphorus availability modulated enzyme activity, with higher phosphatase activities in low-P soils. Furthermore, these findings highlight the importance of root-root interactions and plant species composition in shaping soil biochemical processes.
Protein tyrosine phosphatases have a key catalytic residue on a mobile loop (the WPD-loop), making the connections between this loop sequence and its dynamics, together with the dynamics of other mobile loops, particularly important.
The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.
We report Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid with health-promoting potential. This study was to investigate effects of supplemental DHA from Aurantiochytrium acetophilum on growth performance, health status, meat quality, and protein synthesis signaling of broiler chickens. Day-old male chicks were housed in an environmental control room (6 cages/treatment, 8 chicks/cage), and fed a corn-soybean meal basal diet supplemented with the DHA-rich A. acetophilum biomass (Heliae, Gilbert, AZ) at 0, 1, 2, and 4% (0, 1.7, 3.4 and 6.8 g DHA/kg diet) for 6 weeks. Growth performance was measured weekly. Blood samples were collected at weeks 3 and 6 (2 chicks/cage). Four tissues were sampled (2 chicks/cage) for biochemical and meat quality analyses. Data were analyzed by one-way ANOVA and regression. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) body weight gain (19%) and gain to feed ratio (19%) during weeks 4–6. The A. acetophilum supplementation dose-dependently decreased (p < 0.05, R 2 = 0.21–0.54) plasma alanine amino transferase activity and glucose concentrations, but had little effect on plasma activity of alkaline phosphatase or concentrations of inorganic phosphorus and uric acid at weeks 3 and 6. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) breast muscle weight by 21%, and down-regulated (p < 0.05) mRNA levels of mammalian target of rapamycin and ribosomal s6 protein (S6), and protein levels of phosphorylated S6 to S6 and phosphorylated S6 kinase beta 1 to S6 kinase beta 1. The A. acetophilum supplementation linearly increased (p < 0.01) lipid peroxidation (R 2 = 0.62–0.90) and hardness and chewiness (R 2 = 0.34–0.44) of breast and thigh muscles. In conclusion, supplemental 4% (6.8 g DHA/kg), but not 1 or 2%, of A. acetophilum impaired growth performance, breast muscle mass accumulation, and(or) protein synthesis signaling of broilers.
Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.
Heterotrophic microorganisms in marine sediments produce extracellular enzymes to hydrolyze organic macromolecules, so their products can be transported inside the cell and used for energy and growth. Therefore, extracellular enzymes may mediate the fate of organic carbon in sediments. The Baltic Sea Basin is a primarily depositional environment with high potential for organic matter preservation. The potential activities of multiple organic carbon-degrading enzymes were measured in samples obtained by the International Ocean Discovery Program Expedition 347 from the Little Belt Strait, Denmark, core M0059C. Potential maximum hydrolysis rates (V max ) were measured at depths down to 77.9mbsf for the following enzymes: alkaline phosphatase, β- d -xylosidase, β- d -cellobiohydrolase, N-acetyl-β- d -glucosaminidase, β-glucosidase, α-glucosidase, leucyl aminopeptidase, arginyl aminopeptidase, prolyl aminopeptidase, gingipain, and clostripain. Extracellular peptidase activities were detectable at depths shallower than 54.95mbsf, and alkaline phosphatase activity was detectable throughout the core, albeit against a relatively high activity in autoclaved sediments. β-glucosidase activities were detected above 30mbsf; however, activities of other glycosyl hydrolases (β-xylosidase, β-cellobiohydrolase, N-acetyl-β-glucosaminidase, and α-glucosidase) were generally indistinguishable from zero at all depths. These extracellular enzymes appear to be extremely stable: Among all enzymes, a median of 51.3% of enzyme activity was retained after autoclaving for an hour. We show that enzyme turnover times scale with the inverse of community metabolic rates, such that enzyme lifetimes in subsurface sediments, in which metabolic rates are very slow, are likely to be extraordinarily long. A back-of-the-envelope calculation suggests enzyme lifetimes are, at minimum, on the order of 230days, and may be substantially longer. These results lend empirical support to the hypothesis that a population of subsurface microbes persist by using extracellular enzymes to slowly metabolize old, highly degraded organic carbon.
Increase photorespiration and optimising intrinsic water use efficiency are unique challenges to photosynthetic carbon fixation at elevated temperatures. To determine how plants can adapt to facilitate high rates of photorespiration at elevated temperatures while also maintaining water-use efficiency, we performed in-depth gas exchange and biochemical assays of the C 3 extremophile, Rhazya stricta. These results demonstrate that R. stricta supports higher rates of photorespiration under elevated temperatures and that these higher rates of photorespiration correlate with increased activity of key photorespiratory enzymes; phosphoglycolate phosphatase and catalase. The increased photorespiratory enzyme activities may increase the overall capacity of photorespiration by reducing enzymatic bottlenecks and allowing minimal inhibitor accumulation under high photorespiratory rates. Additionally, we found the CO 2 transfer conductances (stomatal and mesophyll) are re-allocated to increase the water-use efficiency in R. stricta but not necessarily the photosynthetic response to temperature. These results suggest important adaptive strategies in R. stricta that maintain photosynthetic rates under elevated temperatures with optimal water loss. The strategies found in R. stricta may inform breeding and engineering efforts in other C 3 species to improve photosynthetic efficiency at high temperatures.
The effect of soil sterilization by dry heat (0.08% relative humidity), gamma radiation, or both on soil phosphatase, urease, and decarboxylase activity was studied. Soil sterilized by a long exposure to dry heat at relatively low temperatures (eight weeks at 100.5 C) retained higher activities than did soil exposed to a higher temperature (two weeks at 124.5 C), while all activity was destroyed by four days at 148.5 C. Sterilization with 7.5 Mrads destroyed less activity than did heat sterilization. The effect of several individually nonsterizing doses of heat radiation is described.
Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.
We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.
Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.
Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells—a potential cause of neurodegeneration in AOA4 patients.
Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.
Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase toward the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we identified three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are “dual-action” inhibitors that simultaneously block the active site and promote p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38α reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a unique approach to achieving improved potency and specificity for therapeutic kinase inhibitors.
Cells must accurately sense and respond to nutrients to compete for resources and establish growth. Phosphate is a critical nutrient source necessary for signaling, energy metabolism, and synthesis of nucleic acids, phospholipids, and cellular metabolites. During phosphate limitation, fungi import phosphate from the environment and liberate phosphate from phosphate-containing molecules in the cell. In the model filamentous fungus Neurospora crassa, the phosphate starvation response is regulated by the conserved transcription factor NUC-1. The activity of NUC-1 is repressed by a complex of the cyclin-dependent kinase MDK-1 and the cyclin PREG when phosphate is plentiful. When phosphate is limiting, NUC-1 repression by MDK-1/PREG is relieved by the cyclin-dependent kinase inhibitor NUC-2. We investigated the global response of N. crassa to phosphate starvation. During phosphate starvation, NUC-1 directly activated the expression of genes encoding phosphatases, nucleases, and a phosphate transporter and directly repressed genes associated with the ribosome. Additionally, NUC-1 indirectly activated the expression of an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly repressed the expression of genes involved in phosphate acquisition and liberation after an extended period of phosphate starvation. Additionally, NUC-3 directly repressed the expression of the cyclin-dependent kinase inhibitor nuc-2. Thus, through the combination of NUC-3 direct repression of genes in the phosphate starvation response and nuc-2, an activator of the phosphate starvation response, NUC-3 serves to act as a brake on the phosphate starvation response after an extended period of phosphate starvation. This braking mechanism could reduce transcription, a phosphate-intensive process, under conditions of extended phosphate limitation.IMPORTANCEFungi have evolved regulatory networks to respond to available nutrients. Phosphate is often a limiting nutrient for fungi that is critical for many cellular functions, including nucleic acid and phospholipid biosynthesis, cell signaling, and energy metabolism. The fungal response to phosphate limitation is important in interactions with plants and animals. We investigated the global transcriptional response to phosphate starvation and the role of a major transcriptional regulator, NUC-1, in the model filamentous fungus Neurospora crassa. Our data show that NUC-1 is a bifunctional transcription factor that directly activates phosphate acquisition genes, while directly repressing genes associated with phosphate-intensive processes. NUC-1 indirectly regulates an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly represses phosphate acquisition genes and nuc-2, an activator of the phosphate starvation response, during extended periods of phosphate starvation. Thus, NUC-3 acts as a brake on the phosphate starvation response to reduce phosphate-intensive activities, like transcriptional activation, when phosphate starvation persists.
Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme–PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.