Phosphatase Cascade Research Articles

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1–Spo7/Pah1 phosphatase cascade function in lipid synthesis

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

The Phosphatase Cascade Nem1/Spo7-Pah1 Regulates Fungal Development, Lipid Homeostasis, and Virulence in Botryosphaeria dothidea.

Protein phosphatase complex Nem1/Spo7 plays crucial roles in the regulation of various biological processes in eukaryotes. However, its biological functions in phytopathogenic fungi are not well understood. In this study, genome-wide transcriptional profiling analysis revealed that Nem1 was significantly upregulated during the infection process of Botryosphaeria dothidea, and we identified and characterized the phosphatase complex Nem1/Spo7 and its substrate Pah1 (a phosphatidic acid phosphatase) in B. dothidea. Nem1/Spo7 physically interacted with and dephosphorylated Pah1 to promote triacylglycerol (TAG) and subsequent lipid droplet (LD) synthesis. Moreover, the Nem1/Spo7-dependently dephosphorylated Pah1 functioned as a transcriptional repressor of the key nuclear membrane biosynthesis genes to regulate nuclear membrane morphology. In addition, phenotypic analyses showed that the phosphatase cascade Nem1/Spo7-Pah1 was involved in regulating mycelial growth, asexual development, stress responses, and virulence of B. dothidea. IMPORTANCE Botryosphaeria canker and fruit rot caused by the fungus Botryosphaeria dothidea is one of the most destructive diseases of apple worldwide. Our data indicated that the phosphatase cascade Nem1/Spo7-Pah1 plays important roles in the regulation of fungal growth, development, lipid homeostasis, environmental stress responses, and virulence in B. dothidea. The findings will contribute to the in-depth and comprehensive understanding of Nem1/Spo7-Pah1 in fungi and the development of target-based fungicides for disease management.

Conserved regions of the regulatory subunit Spo7 are required for Nem1–Spo7/Pah1 phosphatase cascade function in yeast lipid synthesis

In the yeast Saccharomyces cerevisiae, the Nem1-Spo7 complex is a protein phosphatase that activates Pah1 phosphatidate phosphatase at the nuclear-endoplasmic reticulum membrane for the synthesis of triacylglycerol. The Nem1-Spo7/Pah1 phosphatase cascade largely controls whether phosphatidate is partitioned into the storage lipid triacylglycerol or into membrane phospholipids. The regulated synthesis of the lipids is crucial for diverse physiological processes during cell growth. Spo7 in the protein phosphatase complex is required as a regulatory subunit for the Nem1 catalytic subunit to dephosphorylate Pah1. The regulatory subunit contains three conserved homology regions (CR1, CR2, and CR3). Previous work showed that the hydrophobicity of LLI (residues 54-56) within CR1 is important for Spo7 function in the Nem1-Spo7/Pah1 phosphatase cascade. In this work, by deletion and site-specific mutational analyses, we revealed that CR2 and CR3 are also required for Spo7 function. Mutations in any one of the conserved regions were sufficient to disrupt the function of the Nem1-Spo7 complex. We determined that the uncharged hydrophilicity of STN (residues 141-143) within CR2 was required for Nem1-Spo7 complex formation. In addition, the hydrophobicity of LL (residues 217 and 219) within CR3 was important for Spo7 stability, which indirectly affected complex formation. Finally, we showed the loss of Spo7 CR2 or CR3 function by the phenotypes (e.g., reduced amounts of triacylglycerol and lipid droplets, temperature sensitivity) that are attributed to defects in membrane translocation and dephosphorylation of Pah1 by the Nem1-Spo7 complex. These findings advance knowledge of the Nem1-Spo7 complex and its role in lipid synthesis regulation.

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

Receptor Tyrosine Kinases Mediated Cell Signalling Pathways

A small number of signalling pathways, including receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), are activated by plasma membrane receptors in order for cells to respond to external cues. In addition to transmitting, these pathways also process, encode, and integrate both internal and external signals. In recent years, it has become clear that different spatio-temporal activation profiles of the same set of signalling proteins lead to different gene-expression patterns and various physiological responses. These findings suggest that the precise temporal control and relative spatial distribution of activated signal transducers are necessary for important cellular decisions such as cytoskeletal reorganisation, cell-cycle checkpoints, and cell death (apoptosis). Due to their crucial function in the control of embryogenesis, cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis, RTK-mediated signalling pathways have drawn a lot of attention from scientists. A significant number of serious human diseases, including diabetes, cancer, chronic inflammatory syndromes, and developmental defects, are caused by RTK signalling dysfunction. RTKs undergo allosteric transitions (the insulin receptor, for instance) or dimerization in response to stimulation, which activates the intrinsic tyrosine-kinase activity. Numerous cytoplasmic proteins receive a biochemical signal from subsequent phosphorylation of numerous tyrosine residues on the receptor, which causes them to move to the cell surface. Through intricate biochemical circuits of protein-protein interactions and covalent modification cascades, the resulting cellular reactions take place. The earlier theories of discrete linear pathways, which connected extracellular signals to the expression of particular genes, have been replaced by an emerging picture of interconnected signalling networks, raising concerns about the specificity of signal-response events. In actuality, all RTK-mediated pathways share a common protein complement that mediates signal transduction downstream of RTKs. The expression of nuclear transcription factors is induced by the activation of kinase and phosphatase cascades, such as the mitogen-activated protein kinase (MAPK) cascades, which are both activated by GPCRs and RTKs. There is no single protein or gene that determines the specificity of a signal for any given receptor pathway. Instead, the temporal and spatial dynamics of the components of downstream signalling control specificity. A classic example is the distinctive biological response to EGF and nerve growth factor stimulation of PC12 cell lines (NGF). Cell proliferation is temporarily induced by EGF, whereas cell differentiation is temporarily triggered by sustained MAPK activation by NGF. However, there are many variables that affect how MAPK cascades behave. Depending heavily on their subcellular localization and their recruitment to scaffold proteins, MAPK cascades can produce oscillations, abrupt switches, bistable dynamics.

Phosphorylation-mediated regulation of the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the nuclear/endoplasmic reticulum membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase complex. The phosphorylation of Pah1 by protein kinases, which include casein kinases I and II, Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C, controls its cellular location, catalytic activity, and susceptibility to proteasomal degradation. Nem1 (catalytic subunit) and Spo7 (regulatory subunit), which form a protein phosphatase complex catalyzing the dephosphorylation of Pah1 for its activation, are phosphorylated by protein kinases A and C. In this review, we discuss the functions and interrelationships of the protein kinases in the control of the Nem1-Spo7/Pah1 phosphatase cascade and lipid synthesis.

Phosphatidic Acid Mediates the Nem1‐Spo7/Pah1 Phosphatase Cascade in Saccharomyces cerevisiae

The Nem1‐Spo7/Pah1 phosphatase cascade plays an essential role in the synthesis of the neutral lipid triacylglycerol in the yeast Saccharomyces cerevisiae. Pah1 is a phosphatidic acid (PA) phosphatase that is phosphorylated and inactive in the cytoplasm. The Nem1‐Spo7 complex is a nuclear/endoplasmic reticulum (ER)‐associated protein phosphatase that recruits, dephosphorylates, and activates Pah1. Upon its dephosphorylation, Pah1 hops onto the membrane surface for its interaction with and dephosphorylation of PA to produce the diacylglycerol that is then converted to triacylglycerol. Pah1 then scoots along the membrane to associate with and dephosphorylate another PA molecule. In this work, the Nem1‐Spo7 complex was reconstituted into unilamellar phospholipid vesicles (proteoliposomes) composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and PA to mimic the nuclear/ER membrane. The phospholipid composition of the proteoliposomes, which had an average diameter of 60 nm, was confirmed by thin‐layer chromatography. The reconstituted Nem1‐Spo7 complex supported the recruitment and dephosphorylation of Pah1 phosphorylated by the Pho85‐Pho80 protein kinase complex in vitro or the Pah1 phosphorylated by multiple protein kinases in vivo. The Nem1‐Spo7‐mediated dephosphorylation of Pah1 was dependent on the presence of PA in the proteoliposomes. Additionally, the dephosphorylated Pah1 catalyzed the dephosphorylation of PA to produce diacylglycerol in the proteoliposomes. This reconstituted system permits a defined membrane environment to examine the regulation of the Nem1‐Spo7/Pah1 phosphatase cascade.

Interaction Sites of the Nem1‐Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to produce the diacylglycerol used to synthesize triacylglycerol that is stored in lipid droplets. Pah1 is inactive as a phosphorylated form in the cytosol and becomes active as a dephosphorylated form on the nuclear/ER membrane following its recruitment and dephosphorylation by the Nem1‐Spo7 protein phosphatase complex. Spo7, the regulatory subunit in this protein phosphatase complex, is required for stability and function of the catalytic subunit Nem1. In this work, we examined regions of Spo7 that are involved with the interaction with Nem1 for Nem1‐Spo7 complex formation and interaction with Pah1. By deletion analyses and site‐directed mutagenesis, we found that the Spo7 C‐terminal residues 240‐259 are important for Nem1‐Spo7/Pah1 phosphatase function as indicated by a dramatic reduction in triacylglycerol synthesis and lipid droplet formation, along with a temperature‐sensitive phenotype. We are currently investigating whether this sequence is required for physical interaction with Nem1 or Pah1.

The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Positional Dynamics and Glycosomal Recruitment of Developmental Regulators during Trypanosome Differentiation.

Glycosomes are peroxisome-related organelles that compartmentalize the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite's needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, Trypanosoma brucei PTP1 (TbPTP1), which dephosphorylates and inhibits a serine threonine phosphatase, TbPIP39, which promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream "stumpy" forms to procyclic forms. Detailed microscopy and live-cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, which defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 min of the initiation of differentiation, TbPIP39 relocates to glycosomes, whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a "stumpy regulatory nexus" (STuRN) that coordinates the molecular components of life cycle signaling and glycosomal development during transmission of Trypanosoma bruceiIMPORTANCE African trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies, and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse fly forms is signaled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodeling of their composition and function during the differentiation step, but how this is regulated is not understood. Here we identify a cytological site where the signaling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. In combination, the study provides the first insight into the spatial coordination of signaling pathway components in trypanosomes as they undergo cell-type differentiation.

Lipid droplet biogenesis regulated by the FgNem1/Spo7-FgPah1 phosphatase cascade plays critical roles in fungal development and virulence in Fusarium graminearum.

Lipid droplets (LDs) control lipid metabolism in eukaryotic cells in general. However, the biogenesis regulation and biological functions of LDs are largely unknown in pathogenic fungi. Rapamycin treatment results in a significant increase of LD biogenesis in Fusarium graminearum. Molecular mechanisms of the target of rapamycin (TOR) pathway in regulating LD biogenesis and the functions of LD in virulence of F.graminearum were investigated in depth by combining genetic, cytological and phenotypic strategies. TOR in Fusarium graminearum (FgTOR) inhibition by rapamycin induces LD biogenesis through the FgPpg1/Sit4 signaling branch. FgPpg1 promotes phosphorylation of protein phosphatase FgNem1 by the protein kinase FgCak1. The phosphorylated FgNem1 dephosphorylates the phosphatidate phosphatase FgPah1. Dephosphorylated FgPah1 is active and stimulates LD biogenesis. Moreover, deletion of FgNem1/Spo7 or FgPah1 leads to serious defects in vegetative growth, sexual development and virulence. The results of this study provide novel insights into the regulatory mechanism and biological functions of the LDs in the devastating pathogenic fungus F.graminearum.

An Evolutionarily Conserved Phosphatidate Phosphatase Maintains Lipid Droplet Number and ER Morphology But Not Nuclear Morphology

Phosphatidic acid phosphatases are involved in the biosynthesis of phospholipids and triacylglycerol, and also act as transcriptional regulators. Studies to ascertain its role in lipid metabolism and membrane biogenesis are restricted only to Opisthokonta and Archaeplastida. Herein, we report the role of phosphatidate phosphatase (PAH) in Tetrahymena thermophila, belonging to the distantly related Alveolata clade. We identified two PAH homologs in Tetrahymena, TtPAH1, and TtPAH2. TtPAH1 maintains lipid droplet number and ER morphology but does not regulate nuclear morphology in Tetrahymena. TtPAH1 rescued all the known defects in yeast PAH1 strain and is conserved functionally between Tetrahymena and yeast. The homologous gene derived from Trypanosoma also rescued the defects of yeast PAH1 strain. Our results indicate that PAH1, previously known to be conserved between Opisthokonts, is also present in a set of distant lineages. Thus, a phosphatase cascade is evolutionarily conserved and is functionally interchangeable across eukaryotic lineages.

Phosphorylation of the Nem1‐Spo7/Pah1 Phosphatase Cascade by Pkc1 Protein Kinase C

In the yeast Saccharomyces cerevisiae, the Pah1 phosphatidate phosphatase is responsible for catalyzing the penultimate step in the synthesis of triacylglycerol. Phosphatidate phosphatase dephosphorylates phosphatidate to form diacylglycerol, the precursor of triacylglycerol. Pah1 exists in the cytosol as a phosphoprotein, but it must be dephosphorylated by the Nem1‐Spo7 protein phosphatase for its interaction with the ER membrane to associate with the substrate phosphatidate. In this work, we examined the regulation of the Nem1‐Spo7/Pah1 phosphatase cascade by its phosphorylation by Pkc1 protein kinase C (PKC). Pah1, Nem1, and Spo7 were expressed and purified from Escherichia coli to utilize non‐phosphorylated pristine substrates for PKC, which was purified from yeast. PKC phosphorylated each component of the phosphatase cascade on a serine residue, and the reactions were dependent on phosphatidylserine (0.5 mM) and diacylglycerol (0.15 mM) for the maximum phosphorylations. This was a surprising result given the fact that previous data indicated that yeast PKC does not require lipids for its activity, at least in the absence of GTP‐bound Rho1. Owing that prior work on yeast PKC utilized a peptide substrate, we examined the PKC activity using peptide substrates with sequences derived from Nem1, Spo7, or Pah1. With these substrates, PKC did not require phosphatidylserine and/or diacylglycerol for its activity. We found that mammalian PKC, using Pah1 or the Pah1‐derived peptide as substrates, were dependent on phosphatidylserine and diacylglycerol for activity. These results indicated a fundamental difference in the mechanisms of the reactions catalyzed by the yeast and mammalian PKC enzymes, and that yeast PKC is likely to require lipids for activity in vivo.Support or Funding InformationSupported by NIH grant GM050679

The Nem1‐Spo7/Pah1 Phosphatase Cascade is Negatively Regulated by Protein Kinase A

Yeast Pah1 phosphatidate phosphatase, which dephosphorylates phosphatidate to generate diacylglycerol, catalyzes the penultimate step in the synthesis of triacylglycerol. The translocation of Pah1 from cytosol to ER membrane is essential for its function and is strictly dependent on the nuclear/ER membrane‐associated Nem1‐Spo7 protein phosphatase complex. Dephosphorylation of Pah1 by Nem1‐Spo7 enables its membrane association, and also stimulates its phosphatidate phosphatase activity. Previous work has shown that protein kinase A phosphorylates Pah1 to inhibit its function in lipid metabolism. Here, we showed that Nem1, the catalytic subunit of the protein phosphatase complex, is also phosphorylated by protein kinase A. The kinase phosphorylated Nem1 on multiple serine residues and caused a 35% decrease in its protein phosphatase activity for phospho‐Pah1. The Nem1 synthetic peptides KRNRGSNASEN (Vmax/Km = 2.2 μmol min−1 mg−1 mM−1) and RPRSYSKSELS (Vmax/Km = 94.9 μmol min−1 mg−1 mM−1) that contain Ser‐140 and Ser‐210, respectively, were phosphorylated by protein kinase A. Site‐direct mutagenesis of Ser‐140 and Ser‐210 in Nem1 followed by phosphopeptide analysis showed that Ser‐210 was the major protein kinase A target site. The alanine mutation of Ser‐210 caused a 50% reduction in the in vivo phosphorylation state of Nem1. These data indicated that protein kinase A plays a negative regulatory role on the activity of the Nem1‐Spo7/Pah1 phosphatase cascade.Support or Funding InformationSupported by NIH grant GM050679

An evolutionarily conserved phosphatidate phosphatase maintains lipid droplet number and endoplasmic reticulum morphology but not nuclear morphology.

ABSTRACTPhosphatidic acid phosphatases are involved in the biosynthesis of phospholipids and triacylglycerol, and also act as transcriptional regulators. Studies to ascertain their role in lipid metabolism and membrane biogenesis are restricted to Opisthokonta and Archaeplastida. Here, we report the role of phosphatidate phosphatase (PAH) in Tetrahymena thermophila, belonging to the Alveolata clade. We identified two PAH homologs in Tetrahymena, TtPAH1 and TtPAH2. Loss of function of TtPAH1 results in reduced lipid droplet number and an increase in endoplasmic reticulum (ER) content. It also results in more ER sheet structure as compared to wild-type Tetrahymena. Surprisingly, we did not observe a visible defect in the nuclear morphology of the ΔTtpah1 mutant. TtPAH1 rescued all known defects in the yeast pah1Δ strain and is conserved functionally between Tetrahymena and yeast. The homologous gene derived from Trypanosoma also rescued the defects of the yeast pah1Δ strain. Our results indicate that PAH, previously known to be conserved among Opisthokonts, is also present in a set of distant lineages. Thus, a phosphatase cascade is evolutionarily conserved and is functionally interchangeable across eukaryotic lineages.

Is Ecto-nucleoside Triphosphate Diphosphohydrolase (NTPDase)-based Therapy of Central Nervous System Disorders Possible?

Extracellular nucleotides and nucleosides are signalling molecules acting in all tissues and organs, including the central nervous system (CNS). A wide variety of effects, exerted by ecto-purines, requires that their levels, and ATP in particular, must be precisely controlled. Under physiological conditions, concentration of ecto-purines is regulated by a complex cascade of ecto-enzymes, including ecto-NTPDases (nucleoside triphosphate diphosphohydrolases), ecto-NPPs (nucleotide pyrophosphohydrolases/phosphodiesterases), ectoalkaline phosphatases, and ecto-5'nucleotidase. Adenylate kinase, transferring the phosphate moiety between nucleotides, also plays a role in controlling ecto-purines concentration. Disturbances in the elements of purinergic pathway within the CNS underlie the induction and amplification of many neurological pathologies. ATP released in bulk from the cells, and not degraded by less efficient or dysfunctional ecto-nucleotidases, triggers excitotoxic damage and neuro-inflammation in the brain tissue. High ATP concentration activating specific receptors has been shown to be involved in various disorders throughout CNS, including brain injury and ischemia, neuro-inflammation, epilepsy as well as neuropathic pain and migraine. Taking the above mentioned influence of ATP into consideration, the modulation of ecto-NTPDase activity or its site-targeted delivery seems a good therapeutic method. The availability of effective brain-targeted drug-delivery system is one of the most significant challenges facing potential NTPDase-based treatment of CNS disorders. The application of genetically engineered stem cells as carrier vehicles offers a promising strategy for the efficient delivery of the enzyme to CNS tissues.

Phosphoproteome and proteome analyses reveal low-phosphate mediated plasticity of root developmental and metabolic regulation in maize (Zea mays L.)

Phosphate (Pi) deficiency has become a significant challenge to worldwide agriculture due to the depletion of accessible rock phosphate that is the major source of cheap Pi fertilizers. Previous research has identified a number of diverse adaptive responses to Pi starvation in the roots of higher plants. In this study, we found that accelerated axile root elongation of Pi-deprived maize plants resulted from enhanced cell proliferation. Comparative phosphoproteome and proteome profiles of maize axile roots were conducted in four stages in response to Pi deficiency by multiplex staining of high-resolution two dimensional gel separated proteins. Pro-Q DPS stained gels revealed that 6% of phosphoprotein spots displayed changes in phosphorylation state following low-Pi treatment. These proteins were involved in a large number of metabolic and cellular pathways including carbon metabolism and signal transduction. Changes in protein abundance of a number of enzymes indicated that low-Pi induced a number of carbon flux modifications in metabolic processes including sucrose breakdown and other downstream sugar metabolic pathways. A few key metabolic enzymes, including sucrose synthase (EC 2.4.1.13) and malate dehydrogenase (EC 1.1.1.37), and several signaling components involved in protein kinase or phosphatase cascades, auxin signaling and 14-3-3 proteins displayed low-Pi responsive changes in phosphorylation state or protein abundance. A variety of key enzymes and signaling components identified as potential targets for phosphorylation provide novel clues for comprehensive understanding of Pi regulation in plants. Protein phosphorylation, coordinating with changes in protein abundance, is required for maize root metabolic regulation and developmental acclimation to Pi starvation.

Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin

The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of calreticulin in the regulation of WNT signaling. We show that a deficiency in calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca(2+) buffering capacity of calreticulin, as the protein is important in regulating ER Ca(2+) release and activation of Ca(2+)-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of calreticulin and ER Ca(2+) homeostasis/signaling in the canonical WNT signaling pathway.

Vascular Smooth Muscle Cells activation revealed by quantitative phosphoproteomics analysis

Vascular smooth-muscle cells (VSMCs) are the main components of the artery medial layer and if activated by growth factors (such as PDGF-BB) as a consequence of vessel injuries, acquire the ability to proliferate and migrate contributing to the formation of neointima. In the early times of VSMC stimulation a cascade of kinases and phosphatases initiates phospho-events that are decisive for VSMC activation, which terminates with a reorganization of the cell structure. In this work, a shotgun proteomics approach aimed at disclosing factors involved in these phenotypically imperceptible, but significant events has been applied. A SILAC approach in a multi-strategy combined method for phosphopeptides enrichment was used, in order to gain insights into the phosphomodulation of VSMC proteome stimulated by PDGF-BB. We performed a quantitative SILAC phosphoproteome analysis on primary VSMC cells which allowed the identification of 1300 phosphopeptides among which 47 resulted novel phosphosites. Moreover, in VSMC signaling events, some important factors involved in cytoskeleton remodeling, focal adhesions, gap junction assembly and cell activation have been found differentially phosphorylated, highlighting their pivotal role in early VSMC reorganization and suggesting a novel starting point to assess the precise actors of VSMC activation and their roles.