ThePDE1-encoded Low-Affinity Phosphodiesterase in the YeastSaccharomyces cerevisiaeHas a Specific Function in Controlling Agonist-induced cAMP Signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252into alanine resulted in a Pde1ala252allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Δ. The difference between the Pde1ala252allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2val19pde2Δ background, the Pde1ala252allele caused nearly the same hyperaccumulation of cAMP as pde1Δ, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1ala252mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1ala252enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1ala252, and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

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Thrombospondin-1 promotes haemostasis through modulation of cAMP signalling in blood platelets.

Blood ◽

10.1182/blood.2020005382 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ahmed Aburima ◽

Martin Berger ◽

Benjamin EJ Spurgeon ◽

Beth A Webb ◽

Katie S Wraith ◽

...

Keyword(s):

Platelet Activation ◽

Blood Platelets ◽

Camp Signaling ◽

Camp Accumulation ◽

In Vivo Models ◽

Downstream Signaling ◽

Thrombospondin 1 ◽

Increased Sensitivity

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can promote platelet activation, but its role in haemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of haemostasis that promotes platelet activation by modulating inhibitory cAMP signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of haemostasis and thrombosis demonstrated that TSP-1 deficient mice had prolonged bleeding, defective thrombosis and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild type (WT), but not TSP-1-/- platelets, ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation and arrest under flow by PGI2. Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This suggests that the release of TSP-1 regulates haemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

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Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.5966 ◽

1995 ◽

Vol 15 (11) ◽

pp. 5966-5974 ◽

Cited By ~ 50

Author(s):

M Oelgeschläger ◽

J Krieg ◽

J M Lüscher-Firzlaff ◽

B Lüscher

Keyword(s):

Dna Binding ◽

Phosphorylation Site ◽

Direct Interaction ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Wild Type ◽

Type C ◽

A Site

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

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The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3967 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3967-3979 ◽

Cited By ~ 103

Author(s):

T Tanaka ◽

M Kurokawa ◽

K Ueki ◽

K Tanaka ◽

Y Imai ◽

...

Keyword(s):

Myeloid Leukemia ◽

Phosphorylation Site ◽

Wild Type ◽

Extracellular Signal Regulated Kinase ◽

Biological Phenomena ◽

Extracellular Signal ◽

Dna Binding Affinity ◽

Kinase Pathway

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.

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A Murine FVIIa Variant with Increased Tissue Factor-Independent Intrinsic Activity Corrects the Murine Hemophilia B Phenotype Following AAV Administration: Implications for Therapeutic AAV Vector Doses.

Blood ◽

10.1182/blood.v108.11.3276.3276 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3276-3276

Author(s):

Paris Margaritis ◽

Elise Roy ◽

Katherine A. High

Keyword(s):

Animal Model ◽

Gene Transfer ◽

Tissue Factor ◽

Hemophilia B ◽

Intrinsic Activity ◽

Wild Type ◽

Single Chain ◽

B Mice

Abstract We have previously designed a gene transfer approach using a modified FVII transgene that is cleaved intracellularly, secreted in the active form (FVIIa) and results in phenotypic correction of hemophilia B mice, following adeno-associated virus (AAV)-mediated, liver-directed gene delivery. Research into FVIIa variants with increased catalytic efficiency will not only provide information on the mechanism of action of high-dose FVIIa [tissue factor (TF)-dependent or independent], but may also lower the vector doses required in gene therapy settings. However, the use of such variants in an animal model of hemophilia is lacking. To address this, based on published observations using human FVIIa, we have generated a murine FVIIa variant (L305V/A314E/K337A/I374Y, mFVIIa-VEAY) with substitutions located in the catalytic domain of murine FVIIa. Purified mFVIIa and mFVIIa-VEAY from conditioned medium were almost exclusively in the activated form. Mouse FVII zymogen was purified as a single chain, indicating the lack of autoactivation during purification. Using clotting-based assays for the extrinsic (PT) and intrinsic pathway (aPTT), we found that variant VEAY had almost identical extrinsic activity to unsubstituted mFVIIa but substantially increased intrinsic activity (between 3–7 fold) using FVIII, FIX or FVII deficient plasmas, suggesting that this variant has TF-independent clotting activity. From in vitro kinetic assays, binding to human or murine soluble TF was identical for both mFVIIa and mFVIIa-VEAY (2.0 and 3.2 nM for murine TF, respectively; 86.7 and 71.7 nM for human TF, respectively). Similarly, using the physiological substrate (human FX) at saturating TF conditions, FXa generation was identical for both mFVIIa and mFVIIa-VEAY. However, in the absence of TF, mFVIIa-VEAY exhibited substantial proteolytic activity towards FX (6.6 fold), when compared to mFVIIa (which results in a minimal rate of FXa generation). In rotational thromboelastometry with citrated hemophilia B whole mouse blood and mFVIIa-VEAY or mFVIIa added at a dose of 1μg/ml (corresponding to the clinically effective dose of 90μg/kg), mFVIIa-VEAY exhibited a reduction in the clot time (CT) relative to both mFVIIa-treated hemophilia B blood or wild-type blood [61 ± 3 sec vs. 79 ± 13 sec (mFVIIa-treated) and 106 ± 13 sec (wild-type)]. Lastly, we administered 1.2 × 10^12 vector genomes of AAV8-mFVIIa or AAV-mFVIIa-VEAY into the hepatic circulation of hemophilia B mice. At 2 weeks post transduction, AAV-mFVIIa-VEAY treated mice exhibited superphysiological correction of aPTT and PT, considerably below that of wild type animals (aPTT: 17.4 vs. 28.2 sec; PT: 20.8 vs. 39.7, respectively). In contrast, AAV-mFVIIa treated mice exhibited near normalization of phenotype, indicating that phenotypic correction of the hemophilia B phenotype may be achieved using lower AAV-mFVIIa-VEAY vector doses, compared to mFVIIa. The effect of mFVIIa-VEAY in TF-dependent mouse injury models as well as a low TF mouse background is currently under investigation. In conclusion, we describe a murine FVIIa variant with increased TF-independent activity in vitro; we demonstrate that the variant also shows increased activity in vivo. This variant mFVIIa has potential for lowering the AAV dose in vivo for use in gene transfer applications and may allow for the extension of this approach to a large animal model of hemophilia, since high doses of vector are required to treat hemophilic dogs using the wild-type molecule.

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Yeast AMP Pathway Genes Respond to Adenine through Regulated Synthesis of a Metabolic Intermediate

Molecular and Cellular Biology ◽

10.1128/mcb.21.23.7901-7912.2001 ◽

2001 ◽

Vol 21 (23) ◽

pp. 7901-7912 ◽

Cited By ~ 64

Author(s):

Karine Rébora ◽

Christine Desmoucelles ◽

Françoise Borne ◽

Benoı̂t Pinson ◽

Bertrand Daignan-Fornier

Keyword(s):

Feedback Inhibition ◽

Purine Nucleotide ◽

Wild Type ◽

Metabolic Intermediate ◽

Mutant Strains ◽

Extracellular Purines ◽

Two Hybrid ◽

Intermediate Metabolite

ABSTRACT In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of theADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5′-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.

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Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

Applied Sciences ◽

10.3390/app11156865 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6865

Author(s):

Eun Seon Lee ◽

Joung Hun Park ◽

Seong Dong Wi ◽

Ho Byoung Chae ◽

Seol Ki Paeng ◽

...

Keyword(s):

Cold Stress ◽

Wild Type ◽

Rna Chaperone ◽

E Coli ◽

Cold Sensitive ◽

Thioredoxin H ◽

Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

Journal of Parkinson s Disease ◽

10.3233/jpd-202400 ◽

2021 ◽

pp. 1-24

Author(s):

Juho-Matti Renko ◽

Arun Kumar Mahato ◽

Tanel Visnapuu ◽

Konsta Valkonen ◽

Mati Karelson ◽

...

Keyword(s):

Mapk Signaling ◽

Dopamine Neurons ◽

Dopamine Depletion ◽

Wild Type ◽

Disease Modifying Therapy ◽

Immortalized Cells ◽

Midbrain Dopamine ◽

6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

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The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Nature Communications ◽

10.1038/s41467-021-22861-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sabrina Dietz ◽

Miguel Vasconcelos Almeida ◽

Emily Nischwitz ◽

Jan Schreier ◽

Nikenza Viceconte ◽

...

Keyword(s):

Quantitative Proteomics ◽

Wild Type ◽

C Elegans ◽

Double Stranded Dna ◽

Telomere Sequence ◽

Proteomics Approach ◽

Telomeric Protein ◽

Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

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Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽

10.3390/biomedicines9030320 ◽

2021 ◽

Vol 9 (3) ◽

pp. 320

Author(s):

Thaís Pereira da Silva ◽

Fernando Jacomini de Castro ◽

Larissa Vuitika ◽

Nayanne Louise Costacurta Polli ◽

Bruno César Antunes ◽

...

Keyword(s):

Structural Changes ◽

Capillary Permeability ◽

Structural Data ◽

Histopathological Analysis ◽

Amino Acid Residues ◽

Wild Type ◽

Antigenic Properties ◽

Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

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RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽

10.1093/genetics/166.4.1631 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1631-1640 ◽

Cited By ~ 4

Author(s):

Janet R Donaldson ◽

Charmain T Courcelle ◽

Justin Courcelle

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Dna Lesions ◽

Replication Forks ◽

Wild Type ◽

Replication Machinery ◽

Dna Junctions ◽

Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

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