Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Oct 11, 2013
ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated... more ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated 30% of proteins, making this process one of the most common post-translational modifications. The conformational changes of proteins due to phosphorylation are controlled by the dynamic interaction of protein phosphatases and kinases that constitute signaling pathways in unicellular and multicellular organisms. These signaling cascades control and coordinate a wide variety of intracellular and intercellular processes such as transcription, translation, cell cycle and cell division control, differentiation, motility, and cell–cell and cell–substrate interactions. Each of these processes contributes to the mechanisms that pathogens have evolved to survive in the hostile environment of their hosts and vectors. Consequently, these organisms have acquired a set of signaling molecules, including pathogen-specific kinases and phosphatases, that act within the pathogen itself or interact with the signaling pathways of the vector and the host. One of the most important – and evolutionarily ancient – groups of unicellular parasites is represented by the order of Trypanosomatida. Members of this order are flagellated unicellular organisms, and include extracellular and intracellular parasites responsible for severe diseases in humans and animals, mostly in tropical and subtropical regions of the world. In this chapter, attention will be focused on the protein phosphatases that have been characterized experimentally in the three most important trypanosomatids that infect humans, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.
Comparative biochemistry and physiology. B. Comparative biochemistry, Nov 1, 1992
Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and ... more Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca 2+-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-I is phosphorylated by cyclic AMP-dependent protein kinase.
Biochimica Et Biophysica Acta - Proteins And Proteomics, Oct 1, 1998
The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 ge... more The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52^208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.
Some trypanosomatids multiplying in the phloem sap are responsible for wilts of tropical crops (c... more Some trypanosomatids multiplying in the phloem sap are responsible for wilts of tropical crops (coconut, oil palm) and have a major economic impact in Latin America and the Caribbean. Other trypanosomatids from laticiferous plants appear as “symbionts-like”non pathogenicand others multiply in fruits. Only one arbitrary genus name has been proposed as yet for all these trypanosomatids: “Phytomonas”. For this project we proposed the comparative sequencing of two plant trypanosomatids, one pathogenic isolate from a diseased coconut from Guiana and one latex isolate from Euphorbia.
Protein phosphorylation is one of the most important post-translational modifications regulating ... more Protein phosphorylation is one of the most important post-translational modifications regulating various signaling processes in all known living organisms. In the cell, protein phosphatases and protein kinases play a dynamic antagonistic role, controlling the phosphorylation state of tyrosine (Tyr), serine (Ser) and threonine (Thr) side chains of proteins. The reversible phosphorylation modulates protein function, through initiating conformational changes, which influences protein complex formation, alteration of enzyme activity and changes in protein stability and subcellular localization. These molecular changes affect signaling cascades regulating the cell cycle, differentiation, cell–cell and cell–substrate interactions, cell motility, the immune response, ion-channel and transporter activities, gene transcription, mRNA translation, and basic metabolism. In addition to these processes, in unicellular parasites, like Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., addi...
Glycosomes are peroxisome-related organelles that compartmentalise the glycolytic enzymes in kine... more Glycosomes are peroxisome-related organelles that compartmentalise 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, TbPTP1, that dephosphorylates and inhibits a serine threonine phosphatase TbPIP39 that 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 associa...
Protein phosphorylation is a frequent posttranslational modification regulating cellular processe... more Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and-2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B)-also called calcineurin-is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins.
Protein kinase C (PKC) is a key regulatory element in signal transduction and exerts its effects ... more Protein kinase C (PKC) is a key regulatory element in signal transduction and exerts its effects by catalyzing specific substrate phosphorylation. Today, P K C covers a family of 12 so far known phospholipid-dependent serine-threonine specific protein kinases. PKCdelta, the most thoroughly studied member of the socalled nPKC-subclass, is involved in diverse cellular processes, such as growth, differentiation, apoptosis and tumorigenesis. In order to understand the unique functions of PKCdelta,
The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, prol... more The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of "memory" of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommi...
ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated... more ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated 30% of proteins, making this process one of the most common post-translational modifications. The conformational changes of proteins due to phosphorylation are controlled by the dynamic interaction of protein phosphatases and kinases that constitute signaling pathways in unicellular and multicellular organisms. These signaling cascades control and coordinate a wide variety of intracellular and intercellular processes such as transcription, translation, cell cycle and cell division control, differentiation, motility, and cell–cell and cell–substrate interactions. Each of these processes contributes to the mechanisms that pathogens have evolved to survive in the hostile environment of their hosts and vectors. Consequently, these organisms have acquired a set of signaling molecules, including pathogen-specific kinases and phosphatases, that act within the pathogen itself or interact with the signaling pathways of the vector and the host. One of the most important – and evolutionarily ancient – groups of unicellular parasites is represented by the order of Trypanosomatida. Members of this order are flagellated unicellular organisms, and include extracellular and intracellular parasites responsible for severe diseases in humans and animals, mostly in tropical and subtropical regions of the world. In this chapter, attention will be focused on the protein phosphatases that have been characterized experimentally in the three most important trypanosomatids that infect humans, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1992
Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and ... more Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca 2+-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-I is phosphorylated by cyclic AMP-dependent protein kinase.
Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and pho... more Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.
African trypanosomes cause disease in humans and livestock, generating significant health and wel... more African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.
Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectivel... more Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the innermembrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl 2 mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A ... more Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A, respectively), inhibit Neurospora crassa hyphal growth. When N. cassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the eect of the drug was more pronounced at 34°C than at 25°C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The ®rst, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa. Key words Calyculin A á Cantharidin á Neurospora crassa á Okadaic acid á Protein phosphatase 2A
Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and ... more Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1-5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3,6,7]; each has one β β and several α α isoform genes (α α 1 , α α 2 , α α 3 in flies, α α and γ γ in humans; mammalian PP1β β is also known as PP1δ δ). The α α/β β subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are > >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila β β isoform has a distinct biological role. We show that PP1β β 9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.
African trypanosomiasis is a neglected tropical disease affecting humans and animals across 36 su... more African trypanosomiasis is a neglected tropical disease affecting humans and animals across 36 sub-Saharan African countries. We have investigated the potential to exploit a 'piggyback' approach to inhibit Trypanosoma brucei transmission by targeting the key developmental regulator of transmission, T. brucei protein tyrosine phosphatase 1. This strategy took advantage of the extensive investment in inhibitors for human protein tyrosine phosphatase 1B, a key target for pharmaceutical companies for the treatment of obesity and diabetes. Structural predictions for human and trypanosome tyrosine phosphatases revealed the overall conservation of important functional motifs, validating the potential for exploiting cross specific compounds. Thereafter, nineteen inhibitors were evaluated; seventeen from a protein tyrosine phosphatase 1B-targeted inhibitor library and two from literature analysis-oleanolic acid and suramin, the latter of which is a front line drug against African trypanosomiasis. The compounds tested displayed similar inhibitory activities against the human and trypanosome enzymes, mostly behaving as noncompetitive inhibitors. However, their activity against T. brucei in culture was low, necessitating further chemical modification to improve their efficacy and specificity. Nonetheless, the results validate the potential to explore a 'piggyback' strategy targeting T. brucei protein tyrosine phosphatase 1 through exploiting the large pharmacological investment in therapies for obesity targeting protein tyrosine phosphatase 1B.
Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Oct 11, 2013
ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated... more ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated 30% of proteins, making this process one of the most common post-translational modifications. The conformational changes of proteins due to phosphorylation are controlled by the dynamic interaction of protein phosphatases and kinases that constitute signaling pathways in unicellular and multicellular organisms. These signaling cascades control and coordinate a wide variety of intracellular and intercellular processes such as transcription, translation, cell cycle and cell division control, differentiation, motility, and cell–cell and cell–substrate interactions. Each of these processes contributes to the mechanisms that pathogens have evolved to survive in the hostile environment of their hosts and vectors. Consequently, these organisms have acquired a set of signaling molecules, including pathogen-specific kinases and phosphatases, that act within the pathogen itself or interact with the signaling pathways of the vector and the host. One of the most important – and evolutionarily ancient – groups of unicellular parasites is represented by the order of Trypanosomatida. Members of this order are flagellated unicellular organisms, and include extracellular and intracellular parasites responsible for severe diseases in humans and animals, mostly in tropical and subtropical regions of the world. In this chapter, attention will be focused on the protein phosphatases that have been characterized experimentally in the three most important trypanosomatids that infect humans, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.
Comparative biochemistry and physiology. B. Comparative biochemistry, Nov 1, 1992
Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and ... more Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca 2+-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-I is phosphorylated by cyclic AMP-dependent protein kinase.
Biochimica Et Biophysica Acta - Proteins And Proteomics, Oct 1, 1998
The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 ge... more The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52^208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.
Some trypanosomatids multiplying in the phloem sap are responsible for wilts of tropical crops (c... more Some trypanosomatids multiplying in the phloem sap are responsible for wilts of tropical crops (coconut, oil palm) and have a major economic impact in Latin America and the Caribbean. Other trypanosomatids from laticiferous plants appear as “symbionts-like”non pathogenicand others multiply in fruits. Only one arbitrary genus name has been proposed as yet for all these trypanosomatids: “Phytomonas”. For this project we proposed the comparative sequencing of two plant trypanosomatids, one pathogenic isolate from a diseased coconut from Guiana and one latex isolate from Euphorbia.
Protein phosphorylation is one of the most important post-translational modifications regulating ... more Protein phosphorylation is one of the most important post-translational modifications regulating various signaling processes in all known living organisms. In the cell, protein phosphatases and protein kinases play a dynamic antagonistic role, controlling the phosphorylation state of tyrosine (Tyr), serine (Ser) and threonine (Thr) side chains of proteins. The reversible phosphorylation modulates protein function, through initiating conformational changes, which influences protein complex formation, alteration of enzyme activity and changes in protein stability and subcellular localization. These molecular changes affect signaling cascades regulating the cell cycle, differentiation, cell–cell and cell–substrate interactions, cell motility, the immune response, ion-channel and transporter activities, gene transcription, mRNA translation, and basic metabolism. In addition to these processes, in unicellular parasites, like Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., addi...
Glycosomes are peroxisome-related organelles that compartmentalise the glycolytic enzymes in kine... more Glycosomes are peroxisome-related organelles that compartmentalise 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, TbPTP1, that dephosphorylates and inhibits a serine threonine phosphatase TbPIP39 that 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 associa...
Protein phosphorylation is a frequent posttranslational modification regulating cellular processe... more Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and-2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B)-also called calcineurin-is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins.
Protein kinase C (PKC) is a key regulatory element in signal transduction and exerts its effects ... more Protein kinase C (PKC) is a key regulatory element in signal transduction and exerts its effects by catalyzing specific substrate phosphorylation. Today, P K C covers a family of 12 so far known phospholipid-dependent serine-threonine specific protein kinases. PKCdelta, the most thoroughly studied member of the socalled nPKC-subclass, is involved in diverse cellular processes, such as growth, differentiation, apoptosis and tumorigenesis. In order to understand the unique functions of PKCdelta,
The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, prol... more The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of "memory" of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommi...
ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated... more ABSTRACT In a eukaryotic cell, reversible phosphorylation regulates the functions of an estimated 30% of proteins, making this process one of the most common post-translational modifications. The conformational changes of proteins due to phosphorylation are controlled by the dynamic interaction of protein phosphatases and kinases that constitute signaling pathways in unicellular and multicellular organisms. These signaling cascades control and coordinate a wide variety of intracellular and intercellular processes such as transcription, translation, cell cycle and cell division control, differentiation, motility, and cell–cell and cell–substrate interactions. Each of these processes contributes to the mechanisms that pathogens have evolved to survive in the hostile environment of their hosts and vectors. Consequently, these organisms have acquired a set of signaling molecules, including pathogen-specific kinases and phosphatases, that act within the pathogen itself or interact with the signaling pathways of the vector and the host. One of the most important – and evolutionarily ancient – groups of unicellular parasites is represented by the order of Trypanosomatida. Members of this order are flagellated unicellular organisms, and include extracellular and intracellular parasites responsible for severe diseases in humans and animals, mostly in tropical and subtropical regions of the world. In this chapter, attention will be focused on the protein phosphatases that have been characterized experimentally in the three most important trypanosomatids that infect humans, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1992
Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and ... more Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca 2+-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-I is phosphorylated by cyclic AMP-dependent protein kinase.
Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and pho... more Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.
African trypanosomes cause disease in humans and livestock, generating significant health and wel... more African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.
Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectivel... more Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the innermembrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl 2 mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A ... more Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A, respectively), inhibit Neurospora crassa hyphal growth. When N. cassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the eect of the drug was more pronounced at 34°C than at 25°C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The ®rst, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa. Key words Calyculin A á Cantharidin á Neurospora crassa á Okadaic acid á Protein phosphatase 2A
Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and ... more Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1-5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3,6,7]; each has one β β and several α α isoform genes (α α 1 , α α 2 , α α 3 in flies, α α and γ γ in humans; mammalian PP1β β is also known as PP1δ δ). The α α/β β subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are > >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila β β isoform has a distinct biological role. We show that PP1β β 9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.
African trypanosomiasis is a neglected tropical disease affecting humans and animals across 36 su... more African trypanosomiasis is a neglected tropical disease affecting humans and animals across 36 sub-Saharan African countries. We have investigated the potential to exploit a 'piggyback' approach to inhibit Trypanosoma brucei transmission by targeting the key developmental regulator of transmission, T. brucei protein tyrosine phosphatase 1. This strategy took advantage of the extensive investment in inhibitors for human protein tyrosine phosphatase 1B, a key target for pharmaceutical companies for the treatment of obesity and diabetes. Structural predictions for human and trypanosome tyrosine phosphatases revealed the overall conservation of important functional motifs, validating the potential for exploiting cross specific compounds. Thereafter, nineteen inhibitors were evaluated; seventeen from a protein tyrosine phosphatase 1B-targeted inhibitor library and two from literature analysis-oleanolic acid and suramin, the latter of which is a front line drug against African trypanosomiasis. The compounds tested displayed similar inhibitory activities against the human and trypanosome enzymes, mostly behaving as noncompetitive inhibitors. However, their activity against T. brucei in culture was low, necessitating further chemical modification to improve their efficacy and specificity. Nonetheless, the results validate the potential to explore a 'piggyback' strategy targeting T. brucei protein tyrosine phosphatase 1 through exploiting the large pharmacological investment in therapies for obesity targeting protein tyrosine phosphatase 1B.
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Papers by Balázs Szöor