Phosphodiesterase Activity Research Articles

Understanding the Role of Phosphodiesterase yhjH on Biofilm Formation in PHL628 E. coli

Biofilm serves as a protective mechanism against antimicrobial and disinfectant agents as a means to improve the bacteria's biological fitness. YhjH is a phosphodiesterase that degrades bis-(3’5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) and has been shown to play a role in bacterial stress response by influencing biofilm formation. This temperature dependent phosphodiesterase plays a role in biofilm formation by impacting flagellar, curli, and type 1 pili synthesis via c-di-GMP level modulation. However, the exact mechanism involved in how yhjH impacts biofilm formation is still largely unknown. Here, we investigated how yhjH phosphodiesterase activity affects Escherichia coli biofilm formation by characterizing the influence of endogenous yhjH levels on biofilm formation in comparison to exogenously overexpressed yhjH. Using qPCR, immunoblotting techniques, and biofilm assays, yhjH phosphodiesterase activity was observed in E. coli strain PHL628 at 28 °C and 37 °C. Our initial findings indicate that at both the 48 hour and 72 hour time points biofilm concentration is greater in the bacteria grown in the absence of yhjH overexpression at 28 °C. At 37 °C, the bacteria at both time points with yhjH overexpression have a higher concentration of biofilm. By expanding our understanding of how yhjH phosphodiesterase affects biofilm formation, we could find more effective ways to treat diseases such as cystic fibrosis and Crohn's diseases that are caused by biofilm formation and current global health issues such as antibiotic resistance.

Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Infarction.

Under physiological conditions, cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (also known as GPCRs) with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired.

Identification and characterization of alkaline phosphatase gene phoX in Microcystis aeruginosa PCC7806.

PhoX is an extracellular alkaline phosphatase that is widely found in cyanobacteria and plays an important role in the conversion of extracellular organophosphorus into soluble inorganic phosphorus. However, the phoX gene has not yet been experimentally confirmed to exist in bloom-forming Microcystis species. In this study, we identified a putative phoX gene (GenBank accession no. ARI79942.1) in M. aeruginosa PCC7806 and overexpressed it in Escherichia coli 21 (DE3). The expressed PhoX protein displayed phosphodiesterase and phosphomonoesterase activities. In contrast to other bacterial PhoX proteins, which are activated mainly by Ca2+, Microcysits PhoX was most strongly activated by Mg2+, followed by Co2+, Ca2+, Zn2+ and Mn2+, but it was inhibited by Ni2+. Sequence analysis showed that phoX was highly conserved in the Microcystis genus (DNA similarity > 96% between species). phoX expression responded significantly to different environmental phosphorus levels. When PCC7806 cells were cultured in phosphorus-deficient medium (BG11-P), phoX expression reached its highest level at 2h and then decreased to a low level at 4h. Organophosphate induced the expression of phoX; its expression reached the highest level at 4h and was maintained at a high level at 6h. Our results confirmed a putative phoX gene and demonstrated that the phoX gene of Microcystis is conserved.

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5'-Nucleotidase UshA and the 3'-Nucleotidase CpdB.

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.

Structural and functional studies of SAV1707 from Staphylococcus aureus elucidate its distinct metal-dependent activity and a crucial residue for catalysis

The metallo-β-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55 Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π-π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-β-lactamase fold.

Challenges on Cyclic Nucleotide Phosphodiesterases Imaging with Positron Emission Tomography: Novel Radioligands and (Pre-)Clinical Insights since 2016.

Cyclic nucleotide phosphodiesterases (PDEs) represent one of the key targets in the research field of intracellular signaling related to the second messenger molecules cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). Hence, non-invasive imaging of this enzyme class by positron emission tomography (PET) using appropriate isoform-selective PDE radioligands is gaining importance. This methodology enables the in vivo diagnosis and staging of numerous diseases associated with altered PDE density or activity in the periphery and the central nervous system as well as the translational evaluation of novel PDE inhibitors as therapeutics. In this follow-up review, we summarize the efforts in the development of novel PDE radioligands and highlight (pre-)clinical insights from PET studies using already known PDE radioligands since 2016.

Biochar application under low phosphorus input promotes soil organic phosphorus mineralization by shifting bacterial phoD gene community composition.

Biochar has the potential to enhance microbial-mediated phosphorus (P) cycling in soils, but the underlying mechanisms remain largely unknown. We hypothesized that biochar amendment could enhance the production of acid and alkaline phosphomonoesterase, phosphodiesterase and P mineralization, which may vary depending on the P input. To test this hypothesis, we assessed the impacts of rice straw biochar application (0 and 4%) under different P-input rates (0, 30 and 90kg P ha-1) on the relationships among P fractions, phosphatase activities and alkaline phosphomonoesterase-encoding bacterial (phoD gene) communities in an acidic soil. Biochar application under low P input (< 30kg P ha-1) significantly increased the activities of phosphodiesterase and alkaline phosphomonoesterase but not that of acid phosphomonoesterase and depleted organic P. The results from the structural equation model revealed a dominant role of alkaline phosphomonoesterase in P mineralization. The increase in alkaline phosphomonoesterase activity was not related to an increase in phoD gene abundance but was due to a shift in community composition, which was primarily driven by the soil C:P ratio. Microbial network analysis demonstrated a more complex phoD gene community with more functionally interrelated groups as a result of biochar application under low P input than under high P input. Moreover, the specific enrichment of Micromonosporaceae under C-rich and P-poor conditions may play a critical role in alkaline phosphomonoesterase production and potential P mineralization. In conclusion, we demonstrated that biochar application under low P input supports a more organized phoD gene community and preferentially enriches taxa in terms of their capacity for P mineralization, which in turn may enhance P bioavailability and plant P acquisition.

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

BackgroundThere is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (ICa) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β1-adrenoceptor (β1-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on ICa and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes. MethodsAdult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). ICa was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β1-AR were stimulated with the physiological agonist norepinephrine (100 μM). ResultsIn adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and ICa by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and ICa (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and ICa + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and ICa + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on ICa. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and ICa, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and ICa + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on ICa (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET. ConclusionOur results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller ICa responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.

Phosphodiesterases Expression during Murine Cardiac Development.

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

Silencing of circular RNA circPDE5A suppresses neuroblastoma progression by targeting the miR-362-5p/NOL4L axis

Objective Neuroblastoma (NB) is the most common extra-cranial solid tumour in early childhood. Circular RNAs (circRNAs) have been implicated in the development of NB. The purpose of the current study was to explore the molecular action of circRNA phosphodiesterase 5 A (circPDE5A) in NB malignant progression. Materials and methods The expression levels of circPDE5A, miR-362-5p and nucleolar protein 4 like (NOL4L) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) assay. Cell migration and invasion were evaluated by transwell assay. The levels of glucose consumption and lactate production were measured using the commercial assay kits. Targeted correlations among circPDE5A, miR-362-5p and NOL4L were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assays were performed to examine the role of circPDE5A in tumour growth in vivo. Results Our results revealed that circPDE5A was up-regulated in NB tissues and cells. The silencing of circPDE5A suppressed NB cell proliferation, migration, invasion, and glycolysis in vitro and diminished tumour growth in vivo. Moreover, circPDE5A directly targeted miR-362-5p by binding to miR-362-5p. CircPDE5A silencing impeded NB malignant progression in vitro through up-regulating miR-362-5p. Furthermore, NOL4L was a direct target of miR-362-5p, and NOL4L mediated the regulation of miR-362-5p on NB malignant progression in vitro. Additionally, circPDE5A functioned as a regulator of NOL4L expression via targeting miR-362-5p. Conclusions Our current findings identified that the knockdown of circPDE5A suppressed NB malignant progression at least in part by the regulation of the miR-362-5p/NOL4L axis, providing a novel rationale for developing circPDE5A as a potential target for NB management.

Compartmentation of β2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes.

In cardiac myocytes, cyclic AMP (cAMP) produced by both β1 - and β2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by β1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. The cAMP responses produced by β1 -and β2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. Selective activation of β1 - or β2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. These results support the conclusion that cAMP produced by β2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

The PDE10A Inhibitor TAK-063 Reverses Sound-Evoked EEG Abnormalities in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a genetic neurodevelopmental syndrome characterized by increased anxiety, repetitive behaviors, social communication deficits, delayed language development, and abnormal sensory processing. Recently, we have identified electroencephalographic (EEG) biomarkers that are conserved between the mouse model of FXS (Fmr1 KO mice) and humans with FXS. In this study, we test a specific candidate mechanism for engagement of multielectrode array (MEA) EEG biomarkers in the FXS mouse model. We administered TAK-063, a potent, selective, and orally active phosphodiesterase 10A (PDE10A) inhibitor, to Fmr1 KO mice, and examined its effects on MEA EEG biomarkers. We demonstrate significant dose-related amelioration of inter-trial phase coherence (ITPC) to temporally modulated auditory stimuli by TAK-063 in Fmr1 KO mice. Our data suggest that TAK-063 improves cortical auditory stimulus processing in Fmr1 KO mice, without significantly depressing baseline EEG power or causing any noticeable sedation or behavioral side effects. Thus, the PDE10A inhibitor TAK-063 has salutary effects on normalizing EEG biomarkers in a mouse model of FXS and should be pursued in further translational treatment development.

Phosphodiesterases 2, 3 and 4 can decrease cardiac effects of H2-histamine-receptor activation in isolated atria of transgenic mice

Histamine exerts cAMP-dependent positive inotropic effects (PIE) and positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic mice which overexpress the human H2-receptor in the heart (=H2-TG). To determine whether these effects are antagonized by phosphodiesterases (PDEs), contractile studies were done in isolated left and right atrial preparations of H2-TG. The contractile effects of histamine were tested in the additional presence of the PDE-inhibitorserythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, 1 μM, PDE2-inhibitor) or cilostamide (1 μM, PDE3-inhibitor), rolipram (10 μM, a PDE4-inhibitor), and their combinations. Cilostamide (1 μM) and EHNA (1 μM), rolipram (1 μM), and EHNA (1 μM) and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of histamine to elevate the force of contraction (FOC) in H2-TG. Cilostamide (1 μM) and rolipram (10 μM) alone increased and EHNA (1 μM) decreased alone, and their combination increased the potency of histamine to increase the FOC in H2-TG indicating that PDE3 and PDE4 regulate the inotropic effects of histamine in H2-TG. The PDE inhibitors (EHNA, cilostamide, rolipram) alone did not alter the potency of histamine to increase the heart beat in H2-TG whereas a combination of rolipram, cilostamide, and EHNA, or of rolipram and EHNA increased the potency of histamine to act on the beating rate. In summary, the data suggest that the PCE of histamine in H2-TG atrium involves PDE 2 and 4 activities, whereas the PIE of histamine are diminished by activity of PDE 3 and 4.

Tadalafil modulates intracerebroventricular streptozotocin-induced cognition and memory impairment in the young and middle middle-aged rats

Background: Learning and memory may decline due to Alzheimer’s disease (AD) in older adults. A reduction in cyclic guanosine monophosphate concentration and an increase in phosphodiesterase activity have been reported in the process of aging. Although phosphodiesterase (PDE) type 5 inhibitor, Tadalafil is used to treat erectile dysfunction; PDE inhibitors possibly prevent cognition impairment in aging. This study was designed to investigate the effects of tadalafil on memory in middle-aged and young healthy and AD rats. Methods: Memory impairment was induced by intracerebroventricular (ICV) administration of streptozotocin (STZ; 3 mg/kg) in AD rats. Male Wistar rats (middle-aged and young) were distributed into six groups as follows: two control, two AD, and two AD+tadalafil (1 mg/kg) groups. Saline or tadalafil was administered once a day orally for 40 consecutive days. Animals were tested using novel object recognition (NOR), passive avoidance learning (PAL), and Morris water maze (MWM) tests. Results: Aged AD rats exhibited a significant impairment in cognition in the NOR test and impaired learning and memory in PAL and MWM tests compared with the control aged rats. Tadalafil treatment in aged AD rats significantly improved the discrimination index in the NOR test, decreased the time spent in the dark compartment in the PAL test, and increased time spent in the target quadrant in MWM tests compared with aged AD rats. In young AD rats, treatment with tadalafil significantly enhanced cognition, learning, and memory in the NOR, PAL, and MWM tests compared with young AD rats treated with saline. Conclusion: Tadalafil treatment in aged rats improves cognition and memory after STZ-induced (ICV) memory impairment. It can be concluded that chronic treatment with tadalafil is protective against cognitive, learning, and memory impairment in both young and aged subjects.

Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis.

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3′→5′) GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.

A mutagenic screen reveals NspS residues important for regulation of Vibrio cholerae biofilm formation.

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

A tandem motif-based and structural approach can identify hidden functional phosphodiesterases

Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.

Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes

In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Comparative assessment of soil health indicators in response to woodland and silvopasture land use systems

The change in land-use systems due to new management practices and confined agricultural space leads to a major shift in soil health. Soil microbial community composition, enzyme activities, and soil physiochemical properties are considered as important indicators of soil health. Past research has focused on soil health in response to major land-use systems such as forest, pasture, and cultivated land. However, the response of soil health indicators specifically, biological indicators to silvopasture systems remains understated. Therefore, the objective of our study was to assess the impact of silvopasture (SPS) and woodland (WS) land-use systems on the soil health indicators. The study site consists of 5-years-old southern-pine silvopasture developed from existing woodland and 13-years old southern-pine/hardwood mixed woodland plots. Soil samples were collected and analyzed for microbial analysis using the MiSeq platform next-generation sequencing, enzyme assay using a microplate fluorimetric method, and soil physiochemical properties. Significantly higher soil organic carbon (2.6 ± 0.12%), total nitrogen (0.09 ± 0.002%), Mehlich phosphorous (26.9 ± 3.09 mg kg−1), nitrate (4.8 ± 0.24 mg kg−1), and pH (6.5 ± 0.10) were observed in SPS. SPS had a significantly higher acidic phosphatase (169.5 ± 6.59), alkaline phosphatase (43.6 ± 3.39), phosphodiesterase (10.5 ± 0.71), and β-glucosidase (61.7 ± 3.34) activity, measured in nmol g−1 hr−1, as compared to WS. Proteobacteria, Actinobacteria, and Acidobacteria were the most dominant bacterial phyla while Ascomycota and Basidiomycota were the most dominant fungal phyla in both systems. The relative abundance of Proteobacteria (43.3%) was significantly higher in SPS while that of Acidobacteria (17.5%) was significantly higher in woodland (WS). Similarly, Basidiomycota were dominant (p < 0.0001) in WS while SPS was dominated by Ascomycota (p < 0.0001). Microbial diversity and the microbial community at the class and genus taxonomic levels were also impacted by the SPS. Copiotrophs and phytopathogens were higher in SPS while oligotrophs, decomposers, and ectomycorrhizae were higher in WS. In conclusion, our study found strong evidence that the nutrient input and vegetation composition in southern-pine SPS led to changes in soil physiochemical properties which further caused a shift in soil microbial community composition in these systems identified by principal component analysis.

Identification of a Glycerophosphocholine Phosphodiesterase, GDE5, in chicken

Glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of glycerophosphodiesters to glycerol 3-phosphate and alcohol. It was discovered that the glycerophosphodiesterase family plays a role in lipid metabolism and signal pathway in recent years, but little has been known about the characteristics of chicken GDEs. Here, chicken GDE5 (cGDE5) was identified and characterized for the first time. The full length coding cDNA sequence of cGDE5 was cloned, which encoded a polypeptide with 678 amino acids containing a carbohydrate-binding module 20 (CBM20) and a GDPD domain. Tissue expression profiles showed that cGDE5 mRNA was high in various tissues. such as heart, brain, skeletal muscle and testis. Moreover, cGDE5 was demonstrated to exhibit glycerophosphocholine phosphodiesterase activity. These results together suggested that cGDE5, as a unique member of GDE family, may play multiple roles as a cytoplasmic glycerophosphocholine phosphodiesterase.