Dissertations / Theses on the topic 'Phosphoprotein phosphatases'
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1
Tan, Yves S. H. "Regulation of the type 1 protein phosphatase in saccharomyces cerevisiae." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013031.
2
Wadham, Carol. "Protein Tyrosine Phosphatase Pez : its role in the regulation of cell-cell adhesions." Title page, abstract and table of contents only, 2003. http://web4.library.adelaide.edu.au/theses/09PH/09phw122.pdf.
Abstract:
"March 2003" Bibliography: leaves 206-233. The experimental data presented in this thesis provide evidence that PTP-Pez is an active phosphatase that interacts with and dephosphorylates the adherens junction protein ℓ-catenin. PTP-Pez also associates with proteins that form part of the tight junction complex, the scaffolding protein ZO-1 and the transmembrane protein occludin.
3
Wallis, Lise J., and n/a. "Regulation of Bub1b phosphorylation by protein phosphatase 2A." University of Otago. Dunedin School of Medicine, 2006. http://adt.otago.ac.nz./public/adt-NZDU20070502.114819.
Abstract:
The mitotic spindle checkpoint plays a critical role during the cell cycle by protecting the faithful transmission of chromosomes during mitosis. If chromosomes are improperly bound to the spindle microtubules the checkpoint will prevent progress to anaphase by temporarily arresting cells in metaphase until all the chromosomes are correctly aligned. Bub1b is an essential component of the mitotic spindle checkpoint that transiently localises to kinetochores during mitosis and becomes phosphorylated, a response that is sustained during mitotic arrest. Bub1b has been implicated in other processes related to mitotic progression and is thought to regulate mitotic timing and have a role in caspase mediated cell death after prolonged mitotic arrest. The development of aneuploidy and cancer has been associated with mutations in the BUB1B gene and reduction in the level of Bub1b protein. To further our understanding of Bub1b function in the spindle checkpoint and mitosis, new protein interactions involving Bub1b were identified. This thesis describes the search for alternative proteins that interact with Bub1b, and their function in the mitotic spindle checkpoint and regulation of Bub1b activity.
Using a yeast two-hybrid approach, members of the B56 family of regulatory subunits of serine-threonine protein phosphatase (PP2A) were identified as novel interacting partners of Bub1b. Substrate specificity of PP2A is determined by the regulatory subunits. There are five characterised isoforms of the B56 family, each encoded by separate genes. In addition, some isoforms have several recognised splice variants. Confirmation of interactions by alternative methods demonstrated that the isoforms B56γ and B56[epsilon] preferentially interact with phosphorylated Bub1b, whereas the interaction of the remaining B56 isoforms (α, β and [delta]) occurs at a lower affinity with no specificity for the phosphorylated form. It was further demonstrated that B56γ1 associated with phosphorylated Bub1b in vivo.
Induced overexpression of splice variants of B56γ1 and B56γ2 demonstrated a significant reduction in levels of phosphorylated Bub1b during mitotic spindle checkpoint activation. In addition, an associated lower mitotic index was evident in cells with B56γ1 overexpression. Specific inhibition of PP2A activity with okadaic acid was shown to prolong Bub1b phosphorylation during normal mitosis and to restore the levels of phosphorylated Bub1b in arrested cells over expressing B56γ. These findings suggest a role for PP2A activity in regulation of Bub1b function that is mediated through substrate recognition by B56 regulatory subunits.
4
Cheng, Lina. "Regulation of protein phosphatase 1, PP1 [gamma] 2, in testis/spermatozoa by PPP1R11, PPP1R7 and PPP1R2." [Kent, Ohio] : Kent State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1208813693.
5
Sheppard, Vonda Chantal. "Identification and characterization of diatom kinases catalyzing the phosphorylation of biomineral forming proteins." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/37227.
Abstract:
Diatoms are unicellular photosynthetic algae that display intricately patterned cell walls made of amorphous silicon dioxide (silica). Long-chain polyamines and highly phosphorylated proteins, silaffins and silacidins, are believed to play an important role in biosilica formation. The phosphate moieties on silaffins and silacidins play a significant role in biomineral formation, yet no kinase has been identified that phosphorylates these biomineral forming proteins. This dissertation describes the characterization of a novel kinase from the diatom Thalassiosira pseudonana, tpSTK1, which is upregulated during silica formation. A recombinantly expressed histidine-tagged version of tpSTK1 was capable of phosphorylating recombinant silaffins but not recombinant silacidin in vitro. Through establishing methods for subcellular fraction of T. pseudonana membranes in combination with antibody inhibition assay, it was discovered that native tpSTK1 phosphorylates silaffins but not silacidins in vitro (i.e. it exhibits the same substrate specificity as recombinant tpSTK1). As tpSTK1 is an abundant protein in the ER lumen (~ 0.5 % of total ER protein) it seems highly likely to function as a silaffin kinase in vivo. TpSTK1 lacks clear sequence homologs in non-diatom organisms and is the first molecularly characterized kinase that appears to be involved in biomineralization.
The predicted kinase domain (KD) of tpSTK2, the only T. pseudonana homolog of tpSTK1, was recombinantly expressed and tested for phosphorylation activity. Recombinant tpSTK2-KD and native tpSTK2 exhibited detectable activity with myelin basic protein, but did not phosphorylate silaffins or silacidins in vitro. Western blot analysis demonstrated that native tpSTK2 was not present in the ER, but associated with the cytosol and Golgi membrane containing subcellular fractions.
6
Lee, Gui-in. "Structure and dynamics of the receptor kinase interacting FHA domain of kinase associated protein kinase from arabidopsis." Free to MU campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p3100058.
7
Saraf, Amit Strack Stefan. "Regulation of tyrosine hydroxylase by protein phosphatase 2A." [Iowa City, Iowa] : University of Iowa, 2008. http://ir.uiowa.edu/etd/429.
8
Chakrabarti, Rumela. "Role for PP1 [gamma] 2 in spermatogenesis and sperm morphogenesis." [Kent, Ohio] : Kent State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1176430377.
Abstract:
Thesis (Ph.D.)--Kent State University, 2007.Title from PDF t.p. (viewed Mar. 12, 2009). Advisor: Srinivasan Vijayaraghavan. Keywords: sperm, testes, spermatogenesis, protein phosphatase, knock out, spermatid. Includes bibliographical references (p. 115-129).
9
Sayed, Saba Bilquis. "Studies of the role of phosphoprotein phosphatases in the adrenal cortex." Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300432.
10
Szapiel, Nicolas. "Glc7-E101Q is a novel tool for integrated genomic and proteomic analysis of PP1Glc7 phosphatase functional networks in Saccharomyces cerevisiae." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101656.
Abstract:
Reversible phosphorylation is a major mechanism for regulating the activity, localization and stability of proteins required for vital cellular processes such as glucose metabolism, gene expression, establishment of polarity, mitosis and cytokinesis. Phospho-regulation is driven by the activities of kinases and phosphatases. Together, these enzymes account for ∼3% of eukaryotic genomes and it is estimated that 30% of the eukaryotic proteome is composed of phospho-proteins. Protein kinases (PKs) have been studied extensively, however relatively little is known regarding the signaling networks of protein phosphatases (PPases). The identification of PPase functional networks has been slow due to the redundant nature of the majority of PPases, the complexity of their substrate recognition in vivo, and the lack of large-scale analyses that would facilitate network analysis. We hypothesized that large-scale analysis of genetic interactions using the Synthetic Genetic Array (SGA) and proteomic analyses using 2D-PAGE Difference Gel Electrophoresis (DiGE) could reveal PPase functional networks. Here, we apply this approach to the essential and conserved PP1 PPase Glc7 as it regulates numerous cellular processes in budding yeast. For this study, we created a glc7 hypomorphic mutant (glc7-E101Q) suited for both SGA and DiGE analyses. SGA analysis of glc7-E101Q revealed a broad network of 147 synthetic sick/lethal (SSL) and 178 synthetic rescue (SR) interactions. DiGE comparison of the glc7-E101Q proteome relative to wild-type at medium-resolution (∼1000 proteins) revealed alterations in 39 proteins that changed as a consequence of both the mutation and growth conditions. One of the proteins identified in this analysis was Eno1, a non-essential enolase that is mis-regulated in the presence of glucose and identified a SR mutation in the glc7-E101Q SGA. Subsequent phenotypic analysis suggests a novel, non-metabolic role for Eno1 in the Glc7 interaction network. Our results reveal that parallel analysis, using SGA and DIGE, can reveal novel functions and networks that a single analysis may not detect.
11
Zhao, Xiaotao. "The role of protein phosphatase signaling in the formation of the neuromuscular junction /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202004%20ZHAO.
12
Ng, Wai Sun. "Multi-functions of the PP2A domain of axin /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20NGW.
Abstract:
Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 82-89). Also available in electronic version. Access restricted to campus users.
13
Morris, Erin Rebecca. "FHA domain genes of Arabidopsis /." free to MU campus, to others for purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3144443.
14
Zhou, Jie. "Functions of tyrosine kinases and phosphatases in presynaptic development during neuromuscular junction formation /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202007%20ZHOU.
15
Fan, Wen Jun. "The role of protein phosphatases in myocardial ischaemia and reperfusion." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/21615.
Abstract:
Thesis (MScMed)--Stellenbosch University, 2008.ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential.
AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.
16
Van, Kanegan Michael J. Strack Stefan. "Regulation of nerve growth factor signaling by protein phosphatase 2A." Iowa City : University of Iowa, 2008. http://ir.uiowa.edu/etd/279.
17
Nigavekar, Shraddha S. "Regulation of GLC7 encoded PP1 and analysis of synthetic lethal interactions with ade3 and leu2 in saccharomyces cerevisiae." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013007.
18
Leng, Jie. "Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene." Diss., Virginia Tech, 1994. http://hdl.handle.net/10919/39141.
Abstract:
PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from Sulfolobus solfataricus, from which PP1-Arch was purified over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G- 100), and Mono Q FPLC chromatography. PP1-Arch was identified from the final purified sample by renaturation on an SDS-polyacrylamide gel. The molecular size of PP1-Arch was determined by both gel filtration chromatography and SDS-PAGE as 28 kDa and 33 kDa, respectively, which suggests that PP1-Arch is a monomer. PP1-Arch was found stable at temperatures as high as 90°C. Activation constants for the divalent metal ions Mn²⁺ and Ni²⁺, and the Km for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch in vitro. EDTA, Cu²⁺, Zn²⁺, Pi' and PPi were shown to be inhibitors of PP1-Arch, while many compounds known to affect eukaryotic protein phosphatase activities were found to be without noticeable effect.
N-terminal and an internal peptide sequence of the enzyme were obtained. The gene for PP1-Arch was cloned by a combination of "touchdown" PCR and conventional cloning techniques. The PP1-Arch gene was sequenced on both strands, and the sequence was compared with ones from eukaryotes and bacteriophage λ. The sequence homology between PP1-Arch and PP1/PP2A/PP2B suggests that they belongs to the same genetic family.
A recombinant plasmid which was derived from pT7-7 was constructed for expression of PP1-Arch. The PP1-Arch gene was expressed in E. coli and the activity of the expressed enzyme was tested and shown to be divalent metal ion-dependent. Formation of inclusion bodies of expressed PP1-Arch was demonstrated.Ph. D.
19
Abrivard, Marie. "Caractérisation d'une protéine humaine potentiellement impliquée dans l'infection par Toxoplasma gondii." Paris 7, 2012. http://www.theses.fr/2012PA077127.
Abstract:
Toxoplasma gondii est un protozoaire parasite du phylum des Apicomplexes, à l'origine de la toxoplasmose. T. Gondii est un parasite intracellulaire obligatoire capable d'exploiter les propriétés de l'hôte en manipulant les voies de signalisation, afin de se multiplier ou de persister. Les travaux récents ont mis l'accent sur une famille de kinases parasitaires rapidement sécrétées dans la cellule hôte et qui contrôlent l'expression de nombreux gènes. Au laboratoire, a été identifiée une ser/thr phosphatase 2C parasitaire (TgPP2C) et la première partie de ma thèse met en évidence que des parasites exprimant au locus endogène une version « taggée » de TgPP2C sécrètent l'enzyme dans le cytoplasme au cours de l'infection. Afin d'identifier les partenaires de cette enzyme, un crible double hybride a été conduit et la caractérisation d'un des partenaires potentiels, FM08, a été l'objet de la seconde partie de ma thèse. FM08 est de nature et fonction encore inconnues bien qu'initialement listée comme un potentiel activateur des voies MAPK. Nos données documentent que FM08 lie la TgPP2C et est un partenaire exclusif des filaments intermédiaires dans les cellules épithéliales. FM08 décore en unités plus ou moins discontinues les filaments et le partenariat persiste lorsque les filaments se réorganisent comme lors de la mitose ou après perturbation du potentiel oxydant. Ce partenariat s'élargit avec p38, capable d'interagir avec FM08. Nos travaux actuels visent à comprendre si FM08 est un substrat de p38 et s'il contribue à réguler la réponse du cytosquelette des filaments intermédiaires aux stress et ainsi l'intégrité tissulaire, en dehors ou au cours d'un contexte infectieuxToxoplasma gondii is a protozoan parasite that belongs to the Apicomplexa phylum. It causes toxoplasmosis, a generally benign disease that can turn severe especially in case of immunodeficiency as a consequence of the reaction of parasites that persist in deep tissues. T. Gondii is an obligate intracellular parasite that manipulates the host signaling pathways to build a niche where to grow and persit. To this end, the parasite secretes several proteins, mostly kinases so far, that inhibit innate cellular defense and controls the immune response. We have identified a kinase counteracting protein, namely a ser/thr phosphatase type 2C (TgPP2C). This work shows that parasites modified at the endogenous locus secrete a fusion TgPP2C-HA into the host cell cytoplasm. In order to identify putative TgPP2C partners and substrates, a Yeast Two Hybrid screen has been carried out and has identified FM08 as partner candidate. The second part of this thesis aims at characterizing the FM08 protein for which nothing was known a part from its mention within a list of putative regulators of MAPK pathway. We discover that FM08 binds indeed to TgPP2C but is chiefly is a selective partner of intermediate filaments in epithelial cells. The partnership is quite sustained along the filaments but also when these reorganize into particles as during mitosis or upon oxidative stress. We also point a larger complex including the p38 as a bonafidae partner of FM08. Our current investigation assesses whether FM08 is a substrate of p38 and how it could be involved in controlling the cytokeratin-mediated cell response to stress including during infection in order to preserve cell and tissue integrity
20
Galioot, Amandine. "Contribution à l'étude du rôle des SER/THR protéine-phosphatases PP1/PP2A dans les processus de mort cellulaire et de maturation du précurseur du peptide Beta-Amyloïde." Paris 7, 2013. http://www.theses.fr/2013PA077219.
Abstract:
Notre laboratoire a précédemment proposé un concept appelé "Drug phosphatase Technology" (DPT) basé sur l'utilisation de séquences pénétrantes cationiques capables d'interagir avec les phosphatases PP1/PP2A pour déréguler spécifiquement des signaux intracellulaires. Les Ser. Thr protéine-phosphatases PP1 et PP2A sont des acteurs clé de la signalisation cellulaire et la dérégulation de leur activité via leur interaction avec des partenaires d'origine cellulaire ou virale conduit souvent à de graves dysfonctionnements. Par exemple, la protéine E4orf4 des adénovirus interagit avec PP2A1 et entraîne l'apoptose des cellules infectées ou transformées alors que les cellules saines ne sont pas affectées. Lors de cette étude nous avons identifié une séquence d'interaction entre E4orf4 et PP2A1 à partir de laquelle nous avons caractérisé le peptide DPT-E4orf44, qui provoque l'apoptose de certaines cellules tumorales humaines sans affecter les cellules saines de type fibroblastes. Les protéine-phosphatases de la famille PP1 et PP2A jouent également un rôle important dans la régulation physiologique des substrats neuronaux Tau et APP (précurseur de peptide amyloïde). La diminution de l'activité phosphatase dans les cellules nerveuses entraine une augmentation de l'état de phosphorylation de ces substrats chez les patients atteints de la maladie d'Alzheimer. La deuxième partie de ce travail de thèse a permis de mettre en évidence l'implication les protéine-phosphatases PP1 et PP2A dans la régulation de la phosphorylation du résidu T668 d'APP dont la modification post-traductionnelle est essentielle pour la maturation et le clivage de l'APPOuTlabhas previousry proposed a concept denominated "Drug Phosphatase Technology" (DFT) based on the use of cationic penetrating sequences capable of interacting with PP1/PP2A phosphatases in order to deregulate specific intracellular signais Ser/Thr proteins-phosphatases of PP1 and PP2A family are key factors of the cellular signalization and deregulation of their activity through interaction with cellular or viral protein often leads to severe dysfunctions. For exemple, E4orf4 protein of adenoviruses interacts with PP2A1 and leads to apoptotic death in infected or transformed cells while healthy cells remain unaffected. In a first part of this work, we have identified an interacting sequence between PP2A1 and E4orf4 from which we - have characterized the peptide DFT-F4orf44, which provokes the apoptosis of a subset of human tumoral cellswithout affecting healthy cells of fibroblastic type. Protein-phosphatases of PP1 and PP2A family also play a crucial role in the physiological regulation of neuronal substracts Tau and APP (Amylold Peptid Precursor). A diminution of the phosphatase activity in nerve cells leads to a drastically increasjng of phosphorylation state of these proteins for patients affected by Alzheimer's disease. I The second part of this work has allowed the identification of the phosphatase proteins responsible for the regulation of phosphorylation of APP T668 residue, which is an essential modification for APP maturation and processing
21
Ma, On Ki. "Association of the N-methyl-D-aspartate receptor subunit NR3A with protein phosphatase 2A : structural analysis by site-directed mutagenesis /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202003%20MA.
Abstract:
Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.
22
Lin, Qing. "Molecular cloning, chromosomal mapping and differential expression of type one protein phosphatases in Arabidopsis thaliana /." free to MU campus, to others for purchase, 1996. http://wwwlib.umi.com/cr/mo/fullcit?p9737898.
23
Bielinski, Vincent Anthony. "The role of protein phosphatases in regulation of Drosophila S6 by nutrient signaling pathways." Access to abstract only; dissertation is embargoed until after 5/15/2007, 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=147.
24
Gong, Ping Otsuka Anthony John. "Genetic and biochemical analysis of the interaction between unc-44 AO13 ankyrin and protein phosphatase 2A." Normal, Ill. : Illinois State University, 2005. http://wwwlib.umi.com/cr/ilstu/fullcit?p3196647.
Abstract:
Thesis (Ph. D.)--Illinois State University, 2005.Title from title page screen, viewed September 26, 2006. Dissertation Committee: Anthony J. Otsuka (chair), Radheshyam Jayaswal, Kevin A. Edwards, David L. Williams, Hou Tak Cheung. Includes bibliographical references (leaves 110-124) and abstract. Also available in print.
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Dunn, Jamie D. "Polymer-modified plates for enrichment of phosphopeptides prior to analysis by matrix-assisted laser desorption/ionization mass spectrometry." Diss., Connect to online resource - MSU authorized users, 2007.
26
Stone, Julie Marie. "Interaction of a type 2C protein phosphatase with Arabidopsis receptor kinases /." free to MU campus, to others for purchase, 1996. http://wwwlib.umi.com/cr/mo/fullcit?p9737868.
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Ding, Zhaofeng. "Kinase-interacting FHA domain of kinase associated protein phosphatase phosphopeptide interactions and NMR-detected dynamics /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4729.
Abstract:
Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 24, 2007) Vita. Includes bibliographical references.
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Shouse, Geoffrey P. "Characterization of the functional interaction between two tumor suppressors p53 and B56Gamma-PP2A /." Diss., UC access only, 2009. http://proquest.umi.com/pqdweb?index=31&did=1790085501&SrchMode=1&sid=2&Fmt=7&retrieveGroup=0&VType=PQD&VInst=PROD&RQT=309&VName=PQD&TS=1270138690&clientId=48051.
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Zu, Xin Lin. "Methods for the detection, purification and characterisation of histone H4 histidine kinase and the analysis of protein histidine phosphorylation." University of Western Australia. Biochemistry and Molecular Biology Discipline Group, 2007. http://theses.library.uwa.edu.au/adt-WU2007.0092.
Abstract:
[Truncated abstract] Protein phosphorylation, one of the most important forms of post-translational modification, has been demonstrated to play crucial roles in regulation of cell function. Phosphorylation of protein serine, threonine and tyrosine residues has been the most thoroughly investigated, taking advantage of the acid-stable character of these phosphohydroxyamino acids. Whereas, the cellular occurrence of acid-labile phosphoamino acids, such as phosphohistidine, phosphoarginine and phospholysine was often underestimated due to the acid treatments employed by most of the traditional phosphoamino acid analysis methods. The biological roles of histidine kinases (HKs) in prokaryotes are well understood in contrast to those of HKs in eukaryotes, especially in mammalian cells. However, the evidence has shown that phosphohistidine comprised 6% of phosphoamino acids of the basic nuclear proteins in eukaryotes (Matthews, 1995) and there was more phosphohistidine than phosphoserine in rat liver mitochondria (Bieber and Boyer, 1966). More significantly, phosphohistidine was revealed to be the major phosphoamino acid in phosphorylated histone H4 in regenerating liver in vivo (Chen et al., 1974) and the Walker-256 carcinosarcoma cells in vitro (Smith et al., 1974). Recently, the histone H4 histidine kinase (HHK) activity of human hepatocellular carcinoma (HCC) tumour tissue was measured to be 400 times higher than the normal liver tissue surrounding the tumour. HepG2 cells (HCC cell line) and PIL-2 cells (a p53 knockout mouse tumorigenic liver progenitor cell line) also displayed high HHK activity (Tan et al., 2004). The above observations suggested that HKs and HHKs are playing important roles in both prokaryotes and eukaryotes, including mammals. One major obstacle in the study of HHK study has been the lack of knowledge of the amino acid sequence of an HHK. Attempts at purifying and identifying the HHK from yeast led to the partial purification of a yeast HHK protein(s) at 32kDa (Huang et al., 1991). However, the amino acid sequence of the HHK has not yet been established. ... The success of the separation was demonstrated by the MALDI-TOF-MS and/or ESI-MS spectra of the RP-HPLC fractions. These achievements suggested that it is possible to detect phosphohistidyl histone H4 in vivo using MS under experimental conditions where phosphohistidine is relatively stable. The study in this thesis represents the progression of HHK research in various aspects, including the yeast HHK purification and identification, mammalian HHK partial purification and the methodological developments in detecting histone H4 histidine phosphorylation using MS. Furthermore, new information regarding the physical characteristics of yeast HHKs and its potential role in cellular biology have been documented. It is anticipated that knowledge generated in these studies will contribute to the insight and the understanding of the biological significance of HHK in yeast and mammalian cells.
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Lanser, Brittany. "Characterization of checkpoint adaptation in human fibroblastic glioma cells and an analysis of protein phosphatase inhibitors." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Biological Sciences, c2012, 2012. http://hdl.handle.net/10133/3390.
Abstract:
This thesis reports that checkpoint adaptation occurs in human brain cancer cells. M059K cells, after treatment with camptothecin (CPT), recruited γ-histone H2AX, phosphorylated Chk1 and arrested in the G2 phase. Strikingly, cells escaped the checkpoint, became rounded and entered mitosis as measured by phospho-histone H3 signals. Lamin A/C immunofluorescence microscopy revealed that 48% of the cells that survived checkpoint adaptation contained micronuclei. These data suggest that brain cancer cells undergo checkpoint adaptation and may have an altered genome. This thesis also explored if phosphatases participate in checkpoint adaptation. Human colon cancer cells were treated with CPT and the PP2A inhibitor cantharidin. Following treatment the cells became rounded and 65% were positive for phospho-histone H3 signals indicating that cantharidin caused cells to be in mitosis following CPT treatment. These data suggest that PP2A might have a role in checkpoint adaptation, or participate in a pathway that bypasses checkpoint adaptation.xi, 114 leaves : ill. (some col.) ; 29 cm
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Cosper, Marcus S. "Identification of Myosin Light Chain, Myosin Light Chain Phosphatase, and Rho Kinase in the Corpus Cavernosum of the Rat." Connect to resource online, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1244735791.
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Shakir, Salika Mehreen. "Characterization of a serine/threonine phosphatase-kinase pair in Bacillus anthracis." Oklahoma City : [s.n.], 2010.
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Boy-Rocher, Géraldine. "Le substrat de ERK IEX-1 est un inhibiteur général des phosphatases PP2A de la famille B56 impliqué dans la signalisation TPO." Paris 7, 2008. http://www.theses.fr/2008PA077003.
Abstract:
La voie ERK joue un rôle important dans les fonctions de la TPO. L'équipe avait isolé le gène précoce IEX-1 en tant que substrat de ERK en réponse à la TPO et démontré une régulation réciproque des activités de ces deux protéines : IEX-1 phosphorylée par ERK acquiert une fonction anti-apoptotique ; IEX-1 en liant ERK, maintien son activation. Ce denier effet est du à la capacité de IEX-1 d'inhiber de façon spécifique la famille des phosphatases PP2A contenant les sous-unités régulatrices B56 (PP2A-B56). IEX-1 inhibe PP2A-B56 en permettant la phosphorylation de la sous-unité B56 par ERK ce qui conduit à la dissociation de la sous-unité catalytique de l'enzyme. Ces résultats suggéraient que IEX-1 pourrait séquestrer les sous-unités B56 de PP2A, les empêchant d'agir sur d'autres substrats. En effet, nous avons observé que IEX-1 bloque la déphosphorylation d'Akt sur Ser473 et Thr308 et augmente son activité kinase. Akt est une cible des PP2A-B56. En effet, la surexpression de sous-unités B56 entraîne une diminution de la phosphorylation d'Akt sur Ser4723 et Thr308. L'effet inverse est retrouvé en utilisant les siRNA dirigés contre B56β et γ. Les PP2A-B56 reversent l'effet de IEX-1 sur Akt. En utilisant des mutants dominant-négatifs de ERK et des mutants de phosphorylation de B56, nous avons montré que l'effet de IEX-1 sur Akt est dépendant de la phosphorylation de B56 par ERK. IEX-1 maintien donc l'activation d'Akt et de ERK par le même mécanisme. Ces résultats montrent que IEX-1 agit comme un inhibiteur cellulaire général des PP2A-B56et identifient un nouveau substrat des PP2A-B56 ainsi qu'une nouvelle coopération entre les voies ERK et Akt. Dans les cellules UT7-Mpl, IEX-1 est induit spécifiquement par la TPO, mais pas par l'EPO ou le GM-CSF, et participe au maintien de l'activation de ERK et Akt en réponse à cette cytokine. IEX-1 est aussi exprimée dans les CSH CD34+CD38- puis au cours de la différenciation des mégacaryocytes. Le nombre et la taille des CFC ainsi que la fréquence des LTC-IC sont augmentés lorsque IEX-1 est surexprimée par infection lentivirale dans les cellules CD34+ de sang de cordon ombilical. Des résultats préliminaires montrent que la surexpression de IEX-1 pourrait compenser de façon spécifique l'absence de TPO pour la prolifération des cellules CD34+. Par contre, la surexpression de IEX-1 n'a pas d'effet sur la différenciation mégacaryocytaire. Ces résultats suggèrent que IEX-1 et la famille des PP2A-B56 pourraient jouer un rôle unique dans les capacités de la TPO à maintenir le compartiment des CSHIEX-1 is an early-response gene involved in survival and proliferation, which is rapidly induced by growth factors, viral infections or chemical carcinogens. IEX-1 was previously isolated as a substrate of the kinase ERK and having a dual role in ERK signaling : IEX-1 acquires anti-apoptotic functions upon phosphorylation by ERK and by binding to active ERK, IEX-1 behaves as a positive regulator of ERK activation, prolonging ERK signal in response to various growth factors. We have elucidated the mechanism by which IEX-1 prolong ERK signal. The major Ser/Thr phosphatase involve in ERK activation is the protein phosphatase 2A (PP2A). It is made of three subunits: the structural (A), the regulatory (B) and the catalytic (C) subunit. IEX-1 binds specifically to the B56 regulatory subunits of PP2A. This association enhances B56 phosphorylation by ERK and trigger dissociation from the catalytic subunit leading to the inactivation of the PP2A. We examine whether IEX-1 could control only the dephosphorylation of associated ERK or have a more general effect on other kinase activities controlled by PP2A. By using IEX-1 surexpression and shRNA targetting IEX-1, we found that IEX-1 had no effect on the phosphorylation of MEK, p38 or JNK, but that it encreased and sustained the activation of the kinase Akt by preventing its dephosphorylation both on in residues the308 and ser473. These similar regulations of Akt and ERK activities by IEX-1 prompted us to examine whether the same mechanism operates on the two pathways. Overexpression of IEX-1 and B regulatory subunit together shows that specifically B56 subunit strongly reduced the capacity of IEX-1 to prolong Akt phosphorylation. This indicates that Akt phosphorylation is controlled by PP2A holoenzyme containing B56 subunits. Next we have explored the mechanism by which IEX-1 prevents the activity of B56 on pAkt. To test that, we use an IEX-1 protein mutated in its ERK binding site, loosing its ability to both bind pERK and to inhibit B56-PP2A activity. This mutant was enable to extained the duration of pAKT signal showing that IEX-1 mediated Akt activation is dependent on its capacity to bind ERK. To confirm this result, we expressed IEX-1 together with ERK1 and ERK2 kinase dead mutant, devoided of catalytic activity. Expression of this mutant reduces the capacity to protect Akt from inactivation. This result indicate that the ability of IEX-1 to encreased ERK mediated phosphorylation of B56 subunit provides a general mechanism leading to inhibition of B56 regulatory subunits containing PP2A enzymes function. IEX-1-mediated ERK-dependent inhibition of B56 containing is responsible for its ability to control both ERK and Akt activation
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Vandomme, Audrey. "Caractérisation fonctionnelle du PhosphoTyrosyl Phosphatase Activator chez Plasmodium falciparum : rôle dans la régulation de PP2A et de PP1." Thesis, Lille 2, 2014. http://www.theses.fr/2014LIL2S010/document.
Abstract:
Le paludisme est la première endémie parasitaire mondiale causée par le protozoaire Plasmodium. Cette parasitose est responsable de 219 millions de cas et 660 000 décès par an. La prévalence et la mortalité élevées sont liées notamment à la résistance des parasites aux traitements existants, ce qui rend primordial le développement de nouvelles thérapeutiques. Pour ce faire, une meilleure connaissance de la biologie fondamentale du parasite est nécessaire. Dans ce contexte l’un des axes de recherche concerne la régulation du cycle cellulaire chez Plasmodium et notamment les mécanismes de phosphorylation/déphosphorylation qui sont essentiels.Parmi les nombreux acteurs des mécanismes de phosphorylation, la sérine/thréonine protéine phosphatase de type 2A (PP2A) est, avec PP1, l’une des phosphatases majeures. Cette phosphatase est impliquée dans de nombreux processus cellulaires notamment la mitose, la méiose ou encore l’apoptose. Elle est composée d’une sous-unité catalytique (PP2Ac), d’une sous-unité d’aide à l’agencement spatial (A) et d’une sous-unité régulatrice (B). Il existe quatre familles de sous-unités régulatrices contenant chacune plusieurs membres qui permettent de réguler la localisation, la spécificité et l’activité de PP2A. Il existe également des protéines régulatrices indépendantes, notamment les inhibiteurs 1 et 2, la protéine α4 et le PhosphoTyrosyl Phosphatase Activator (PTPA). Chez Plasmodium falciparum, la protéine phosphatase de type 2A ou PfPP2A a été identifiée et semble essentielle pour le développement asexué du parasite. Cependant, peu de choses sont connues sur sa régulation chez le parasite. En effet, seul l’inhibiteur 2 de PP2A a été décrit et caractérisé. Au cours de cette thèse, nous avons effectué par des études in silico un recensement des régulateurs putatifs de PfPP2A. Ces études nous ont permis d’identifier la sous-unité A et une unique sous-unité B. Parmi les régulateurs spécifiques, outre l’inhibiteur 2 déjà caractérisé, l’analyse du génome du parasite montre qu’il contient un orthologue de l’inhibiteur 1, d’α4 et de PTPA. Le projet de cette thèse s’articule autour de la caractérisation moléculaire et fonctionnelle de l’un de ces régulateurs : PfPTPA.La caractérisation moléculaire de PfPTPA a permis de montrer dans ce travail la conservation de cette protéine au cours de l’évolution. L’analyse de sa séquence a révélé que cinq des six motifs de fixation à la PP2A identifiés chez l’homme sont conservés. Par des études in vitro et in vivo dans un modèle hétérologue, nous avons pu confirmer le rôle d’activateur de PfPTPA vis-à-vis de la PP2A. Par une approche de mutation unique d’acides aminés, nous avons identifié trois résidus impliqués dans l’interaction et l’activité de PfPTPA notamment le résidu G292 qui est essentiel pour l’interaction PfPTPA/PfPP2A. Nous avons ensuite montré par des études de génétique inverse que PfPP2A et PfPTPA, qui sont présents dans le même compartiment cellulaire au cours du cycle érythrocytaire, sont essentielles pour la complétion du cycle intra-érythrocytaire du parasite. De plus, PfPTPA semble impliqué dans le cycle cellulaire chez le xénope.En parallèle, l’analyse de la séquence de PfPTPA, a révélé la présence, spécifique au parasite, d’un motif de fixation à la PP1 (motif RVxF). L’identification de ce motif, nous a incités à étudier la relation entre PfPTPA et PfPP1. Nous avons ainsi pu montrer que PfPTPA était capable de se lier à PfPP1 même si elle est incapable de réguler son activité.L’ensemble de ce travail de thèse a permis de caractériser chez Plasmodium falciparum un activateur de la protéine phosphatase de type 2A et de montrer sa spécificité par rapport à la protéine humaine. Nos résultats, et notamment l’implication de PfPTPA dans la régulation du cycle cellulaire, font de ce régulateur une cible thérapeutique potentielleMalaria is the most deadly parasitic disease in the world caused by the Apicomplexa protozoan Plasmodium falciparum. This parasite is responsible for 219 million cases and 660 000 deaths per year and the drug resistance increases the prevalence and the morbidity. The emergence of multi-drug resistance requires the development of new therapeutics. Hence, a better understanding of parasitic fundamental biology is necessary. In this context, one research axis is the cell cycle regulation of Plasmodium, notably phosphorylation/dephosphorylation mechanisms which are essential for the parasite.Among the actors of the reversible phosphorylation, the serine/threonine phosphatase type 2A (PP2A) in eukaryote is, with PP1, one of the major phosphatases. It is involved in several cell processes like mitosis, meiosis or apoptosis. PP2A is composed of a catalytic subunit (PP2Ac), a scaffold subunit (A) and a regulatory subunit (B). There are four regulatory subunit families which regulate location, specificity and activity of PP2A. Furthermore, several independent regulatory proteins including inhibitor 1 and 2, the α4 protein or the phosphotyrosyl phosphatase activator (PTPA) were identified.In Plasmodium falciparum, the protein phosphatase type 2A named PfPP2A has been characterized and seems to be essential for the parasite asexual development as shown by the inhibition of parasitic growth after treatment with natural toxins inhibiting phosphatases. However, its regulation is still poorly understood in Plasmodium. Indeed, only the PP2A inhibitor 2 is characterized in P. falciparum and in P. berghei (a rodent specific Plasmodium species). Using an in silico study, we have identified a putative scaffold subunit and only one B subunit. Among the regulatory proteins, we have identified orthologs of the inhibitor 1, α4 and PTPA. The purpose of this thesis is to study PfPTPA both of the molecular and functional levels.The molecular characterization of PfPTPA showed the evolutionary conservation of this protein. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human, are conserved in P. falciparum. In vitro binding and functional studies revealed that PfPTPA binds to and activates PfPP2A. Mutation studies showed that three residues (V283, G292 and M296) of PfPTPA are indispensable for the interaction and that G292 residue is essential for its activity. Localization studies indicated that PfPTPA and PfPP2A are localized in the same cellular compartment throughout the erythrocytic cycle of P. falciparum, suggesting a possible interaction of both proteins in vivo. In Plasmodium falciparum, genetic studies likely suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Functionnal studies, using Xenopus oocyte, showed that PfPTPA blocked the G2/M transition. Further analysis of PfPTPA sequence revealed that PfPTPA, unlike its human counterpart, possess one of the most canonical binding motif to PP1 (RVxF motif). The identification of this RVxF motif led us to study the role PfPTPA on PfPP1. Thus, we have shown that PfPTPA interacts with PfPP1 but was unable to regulate PfPP1 activity in vitro. This work allowed characterizing the PfPTPA, an activator of protein phosphatase type 2A in Plasmodium falciparum and to show some specificities when compared to its human ortholog. Our data which suggest that this regulator could be involved in cell cycle regulation, together with its essentiality for the growth of P. falciparum strongly support the idea to explore it as potential drug target
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Godet, Angélique. "Contribution à l'étude du rôle de la phosphatase PP2A1 dans l'apoptose induite par la protéine Vpr du VIH." Paris 7, 2009. http://www.theses.fr/2009PA077211.
Abstract:
Le virus de l'immunodéficience humaine de type 1 (VIH-1) est responsable du Syndrome d'ImmunoDéficience Acquise (SIDA). Vpr; une petite protéine auxiliaire de 96 acides aminés (14 kDa) est incorporée dans le virion et joue un rôle important dans l'infection in vivo et dans la pathogenèse du SIDA. Vpr est une protéine pénétrante qui peut être retrouvée libre, sous forme de protéine entière ou de fragments peptidiques, dans le sérum et le liquide céphalorachidien des patients infectés. Vpr peut induire la mort de différentes lignées humaines et de lymphocytes primaires, en pénétrant dans ces cellules. Au court de cette étude, nous avons caractérisé un court domaine pénétrant de la protéine Vpr du VIH-1 (Vpr₇₇₋₉₂). Il présente une variabilité de séquence en fonction des isolats viraux et seules certaines d'entre elles interagissent avec PP2A₁ et induisent une voie. De signalisation apoptotique. Nous avons identifié deux principaux acteurs : PPlc et AIF qui sont impliqués dans la voie de signalisation responsable de cette apoptose. Vpr, avec d'autres protéines du VIH-1, semblent à l'origine de troubles neurocognitifs associés au VIH. Or PP2A₁ est connue pour son implication dans les tauopathies. De plus, AIF (Apoptosis Inducing Factor) est connu comme'facteur apoptotique important dans l'apoptose neuronale. Les cellules neuronales pourraient donc être plus sensibles que les autres types cellulaires à la voie de signalisation apoptotique induite par l'interaction de Vpr avec PP2Ax. De plus nous avons identifié sur AIF une courte séquence d'interaction avec PP₁ : AIF₅₆₆₋₅₇₂ qui interagit avec la protéine PPlc et est un domaine pro-apoptotiqueHuman Immunodeficiency Virus type 1 (HIV-1) encodes several auxiliary proteins including Vpr, a short protein (14kDa). HIV-1 Vpr is a virion-associated protein and interestingly the protein or fragments of Vpr are found in biological fluid such as blood and cerebrospinal fluid. HIV-1 Vpr is a penetrating protein that can cause cell death in both infected and non infected cells. This capacity of Vpr to induce apoptosis in neuronal cells suggests its role in HAND (HIV Assiociated Neurocognitive Disease). Recently we proposed a new concept named Drug Phosphatase Technology, "DPT" used to deregulate intracellular survival pathways and to interfere specifically with the signaling properties of PP1/PP2A family of protein-phosphatases. Consistently with the DPT concept, which is based on the rational design of PP1/PP2A interacting peptides, we have initially identified a PP2A₁docking sequence in the C-terminal portion of HIV-1 Vpr which encompasses amino acid residues 77-92. This domain named Vpr₇₇₋₉₂ interacts both in vitro and in cell extracts with the structural A subunit and with the trimeric ABαC holoenzyme named PP2A₁. Other results suggest that the Vpr₇₇₋₉₂ domain could also be a penetrating apoptotic domain. Therefore we seeked for the role of PP2 in apoptotic pathways mediated by the penetrating sequence Vpr₇₇₋₉₂ in human HeLa and Jurkat cells. We also identified two apoptotic partners: AIF (Apoptosis Inducing Factor) and PP1c. This apoptotic pathway could be particularly important in neuronal survival pathways, and also be contributing at the HAND
36
Habchi, Johnny. "Flexibilité au sein de la nucléoprotéine et de la phosphoprotéine des Paramyxovirus : prédiction, caractérisation expérimentale et repliement induit." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4091.
Abstract:
Les virus Nipah (NiV) et Hendra (HeV) appartiennent au genre Henipavirus au sein de la famille des Paramyxoviridae. Cette famille comporte de nombreux pathogènes tel que le virus de la rougeole (MeV). Les paramyxovirus possèdent un génome de type ARN simple brin encapsidé par la nucléoprotéine (N) au sein d'une nucléocapside hélicoïdale. N interagit avec la phosphoprotéine (P) et cette dernière recrute la polymérase (L) qui assure la transcription et la réplication du génome viral. L'objectif de mon projet de thèse était de caractériser les protéines N et P ainsi que les interactions qui existent entre elles chez les trois virus, NiV, HeV et MeV. A la différence du MeV, qui a été intensivement étudié au cours des dernières années, les données moléculaires et structurales sur les Henipavirus étaient très limitées. A l'aide d'analyses computationnelles, nous avons pu déchiffrer l'organisation modulaire de N et de P, et nous avons montré que les régions, C-terminale de N (NTAIL) et N-terminale de P (PNT), sont prédites comme intrinsèquement désordonnées (RIDs). Les RIDs sont des régions fonctionnelles dépourvues de structures secondaires et tertiaires stables dans des conditions physiologiques. En utilisant des approches biochimiques et biophysiques, nous avons confirmé que NTAIL et PNT sont désordonnées. Elles conservent toutefois des structures secondaires transitoires qui pourraient correspondre à des éléments de reconnaissance moléculaire (ou MoREs) impliqués dans de transitions structurales en présence d'un partenaireThe Paramyxoviridae family includes many important human and animal pathogens, such as measles virus (MeV), a morbillivirus, and the emerging Nipah (NiV) and Hendra (HeV) viruses, members of the Henipavirus genus. Paramyxoviruses possess a negative-strand RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. N interacts with the phosphoprotein (P), and this latter recruits the polymerase that ensures genome replication and transcription. My PhD project has mainly focused on the characterization of the N and P proteins and on the interactions between these two proteins from the three cognate viruses, namely NiV, HeV and MeV. While MeV has been extensively studied through the past years, structural and molecular information on Henipavirus N and P proteins were rather scarce. Using computational analyses, we deciphered the modular organization of Henipavirus N and P. Intrinsically disordered regions (IDRs) were predicted within these proteins, notably at the C-terminus of N (referred to as NTAIL), and at the N-terminus of P (referred to as PNT). IDRs are functional despite they lack of a well-defined 3-D structure under physiological conditions. Biochemical and biophysical approaches pointed out a mostly disordered state for both NTAIL and PNT, although they were shown to contain short-order prone segments (i.e. molecular recognition elements, MoREs). These latter are involved in partner recognition and in disorder-to-order transitions. The C-terminal domains of the P proteins (referred to as PXD) were found to bind to NTAIL and to induce an α-helical transition thereof
37
Blocquel, David. "Etudes biochimiques et biophysiques des protéines de la machinerie réplicative des paramyxovirus." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4110.
Abstract:
Les virus Nipah (NiV) et Hendra (HeV) sont des paramyxovirus zoonotiques appartenant au genre Henipavirus. Les paramyxovirus possèdent un génome ARN simple brin de polarité négative encapsidé par la nucléoprotéine (N) au sein d’une nucléocapside hélicoïdale. Cette dernière sert de substrat pour la transcription et la réplication, réalisées par la polymérase virale qui consiste en un complexe entre la protéine L et la phosphoprotéine (P). A l’aide d’approches biophysiques, j’ai établit une cartographie de l’interaction entre la région C-terminale désordonnée de N (NTAIL) et la région C-terminale de P (PXD) chez NiV, HeV et MeV. L’observation à l’échelle atomique par RMN a confirmé l’intervention d’un élément de reconnaissance moléculaire (MoRE) qui subit un repliement α-hélical au contact de PXD. J’ai également montré la capacité des domaines NTAIL et PXD des henipavirus à former des complexes hétérologues soulignant leur proximité structurale. L’interaction NTAIL-PXD, cruciale pour le recrutement de la polymérase virale constitue une cible idéale pour des approches antivirales. Ainsi, un test de criblage à haut débit par HTRF a été mis en place dans le but d’identifier des inhibiteurs. Enfin, une approche structurale a révélé une organisation trimérique de la protéine P de NiV et HeV en solution. La résolution de la structure cristalline de la région de tétramérisation de P du virus de la rougeole montre la présence d’une région désordonnée à proximité du site putatif de recrutement de L. Collectivement, ces résultats représentent une étape clé vers l’élucidation du l’impact fonctionnel de l’oligomérisation de la protéine P sur le cycle réplicatif des paramyxovirusNipah (NiV) and Hendra (HeV) viruses are zoonotic paramyxoviruses that belong to the Henipavirus genus. Paramyxoviruses possess a single-stranded negative-sense RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. This latter is the substrate for both transcription and replication that are carried out by the polymerase, consisting of a complex between the large protein (L) and the phosphoprotein (P). Using various biophysical approaches, I was able to map the interaction between the C-terminal disordered region of N (NTAIL) and the C-terminal region of P (PXD) in NiV, HeV and MeV. Atomic resolution description of the HeV NTAIL-PXD interaction by NMR confirms the involvement of a molecular recognition element (MoRE) of α−helical nature in binding to PXD. I also showed that Henipavirus NTAIL-PXD form heterologous complexes, involving a structural similarity. As this interaction is crucial for the recruitment of the viral polymerase, it is a promising target for antiviral approaches. This prompted me to set up a protein-protein interaction (PPI) assay based on the HTRF technology to identify inhibitors. Finally, I provided the first experimental evidence of a trimeric organization of P proteins in NiV and HeV. We also solved the crystal structure of two different forms of MeV P tetramerization domain who unveiled the presence of a disordered region located near the putative L-binding site and reveal significant structural variations in coiled-coils organization. Collectively, these results represent a key step towards the elucidation of the functional impact of P protein oligomerization on the replicative cycle of paramyxoviruses
38
Yabukarski, Filip. "Etudes structurales du complexe de réplication des Rhabdoviridae et des Paramyxoviridae. Les interactions entre la phosphoprotéine et la nucléoprotéine." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV033/document.
Abstract:
Le virus de la stomatite vésiculaire (VSV) et le virus Nipah (NiV) appartiennent respectivement aux familles des Rhabdoviridae et des Paramyxoviridae. VSV est un modèle du virus de la rage tandis que NiV est un virus émergeant, appartenant à la sous-famille des Paramyxovirinae, pour lequel les données moléculaires et structurales sont limitées. Ces sont des virus enveloppés dont le génome code pour cinq à neuf protéines. Le complexe de réplication de ces virus est constitué de trois protéines : la phosphoprotéine (P), la nucléoprotéine (N) et la polymérase virale (L). La N encapside le génome viral et l'ensemble N-ARN sert de matrice pour la transcription et la réplication. La P joue deux rôles : elle sert de cofacteur pour la polymérase et forme le complexe N0-P qui maintient la N sous une forme soluble, compétente pour l'encapsidation des génomes néo-synthétisés. Un premier objectif de mon travail de thèse consistait à étudier la structure et la dynamique des protéines P de VSV et de NiV. Ce sont des protéines modulaires qui contiennent des domaines structurés, séparés par des régions flexibles. A mon arrivée au laboratoire un travail important avait été déjà réalisé sur la P de VSV et j'ai participé à l'achèvement de cette étude. Je me suis ensuite intéressé à la protéine P de NiV. J'ai cristallisé et résolu par diffraction des rayons X les structures du domaine C-terminal et du domaine central (codes PDB : 4F9X et 4GJW). La combinaison de ces modèles cristallographiques avec des données de SAXS sur la P entière et des données de résonance magnétique nucléaire (RMN, collaboration IBS) va permettre d'obtenir un modèle atomique de la P entière sous la forme d'un ensemble de conformères. Un deuxième objectif était d'étudier les complexes N0-P. J'ai activement participé au développement de la méthode de reconstitution et à la caractérisation structurale du complexe N0-P de VSV, entre un mutant de la N (NΔ21) et un peptide N-terminal de la P (code PDB : 3PMK). J'ai ensuite reconstitué, cristallisé et résolu la structure de complexe N0-P de NiV entre la N (tronquée de son domaine C-terminal) et la partie N-terminale de la P. Ces structures montrent par quel mécanismes moléculaires la P maintien la N sous forme monomérique, en empêchant sa polymérisation et son interaction avec l'ARN. Les résultats présentés ici ont permis de générer de nouvelles hypothèses pour expliquer les mécanismes d'encapsidation et d'initiation de la synthèse d'ARN chez ces virus. Le complexe N0-P étant essentiel pour la réplication du virus, l'information structurale obtenue au cours de ce travail devrait permettre d'envisager l'utilisation de ce complexe comme cible pour le développement de composés antivirauxAbstract Vesicular stomatitis virus (VSV) and Nipah virus (NiV) belong to the Rhabdoviridae and Paramyxoviridae families, respectively. VSV serves as model system for rabies virus while NiV is an emerging pathogen of the Paramyxovirinae subfamily, for which molecular and structural data are scarce. Both viruses are enveloped and their genomes encode five to nine proteins. Three proteins form their replication complex: the phosphoprotein (P), the nucleoprotein (N) and the viral polymerase (L). N encapsidates the viral genome and this N-RNA complex serves as template for transcription and replication. P has two functions: it serves as a polymerase cofactor and forms an N0-P complex, which keeps the N protein in a soluble and monomeric state, competent for the encapsidation of the newly synthesized genomes. The first goal during the PhD work was to study the structure and dynamics of the VSV and NiV P proteins. These proteins are modular, containing structured domains separated by flexible regions. Before my arrival, a large amount of work was already done on the VSV P protein in the lab and I was involved in the final stages of this work. Then this I studied the NiV P protein, crystallizing and solving the structures of its Central and C-terminal domains by X-ray crystallography (PDB codes: 4F9X and 4GJW). Combining these structures with small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR, collaboration with IBS group) data obtained for the entire protein will allow the construction of an atomic model of the phosphoprotein in the form of a conformational ensemble. The second goal was to study the N0-P complex. I actively participated in the development of the method which permitted the reconstruction of the VSV N0-P complex, using a truncation mutant of the N protein (NΔ21) and an N-terminal peptide from P, and to its structural determination (PDB code: 3PMK). Then I reconstructed, crystallized and solved the structure of the NiV N0-P complex using a C-terminally truncated N protein and the N-terminal region of the P protein. Both structures yielded insights into the molecular mechanisms used by the phosphoproteins in order to maintain the corresponding nucleoproteins in their monomeric state, thus inhibiting their polymerization and interaction with RNA. The results presented here also offered new hypothesis about mechanisms of encapsidation and of RNA synthesis initiation. Given that the N0-P complex is an essential component of the replication complex, the structural information gained from this work allow us to consider this complex as a potential antiviral target
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Kim, Seung-jae. "Studies of HTLV-1 p12(I) in calcineurin binding, calcium-mediated cell signalling and viral transmission." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1148487909.
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Schramm, Antoine. "Caractérisation de domaines d’oligomérisation et régions désordonnées de phosphoprotéine de paramyxovirus." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0381.
Abstract:
Chez les paramyxovirus, la réplication viral est gouvernée par le complexe réplicatif. Composée des protéines N, P et L, il constitue une cible de choix pour des inhibiteurs anti-viraux. La P est caractérisée comme la plateforme de recrutement principale unissant les différents éléments du système. Cependant, l’interaction entre la P et la L reste peu caractérisée. L’objectif de ma thèse est d’améliorer d’une part la cartographie de P en rapport avec son interaction avec L et d’autre part d’améliorer les modèles structuraux de P. En utilisant différentes approches nous sommes parvenus à identifier différents éléments de la Pinter agissant avec la L et à associer une fonction à ces interactions. Notamment, le module P XD combiner à un domaine d’oligomérisation est indispensable pour former un complexe stable et soluble entre P et L. Nous avons identifié un domaine en C-ter du domaine d’oligomérisation dont la présence est indispensable pour le repliement de L sous forme active. Finalement, nous avons mis en évidence qu’une propriété physico-chimique du domaine d’oligomérisation est crucial pour le fonctionnement de la machinerie de transcription et réplication virale. Des mesures de biophysiques sur des fragments de P permirent de proposer un modèle préliminaire de la structure du domaine C-terminale de P à très basse résolution.La cartographie de l’interaction entre P et L suggère que ces deux protéines interagissent via différents sites, suggérant un complexe dynamique au sein duquel le domaine d’oligomérisation, jusqu’ alors considéré comme un domaine relativement inerte, jouerait un rôle crucial dans la machinerie de transcription et réplicationThe replication of paramyxoviruses is headed by the viral replicative machinery composed of three proteins : P, N and L.This complexe is a promising target for antiviral inhibitors. While P is known to be the centralrecruitment platform of the system, the interaction details of the P-L complex remain obscure. The ambition ofmy thesis project is to improve our knowledge on P interaction and dynamics. Thus, on one hand my goal is toimprove the P-L interaction mapping and on the other hand to contribute to a better structural description of P.Combining bio-physical and functional virology approaches, we identified P modules involved in the P-Lcomplex assembly. P XD is an essential P module for interaction as well as the P oligomeric state. theoligomerisation domain C-terminal part is essential for P chaperon function and crucial to drive L to an activeconformation. Finally, physico-chemical properties that correlate the oligomerisation domain stability is essentialfor transcription and replication processes. Moreover, based on biophysical measurements on P truncatedvariants, we propose a preliminary model of P C-terminal domain at very low resolution. Those results are a stepforward in narrowing down the replicative complex mechanistic. P mapping suggest that P and L interacttogether via different sites, suggesting this complex is rather dynamic. Especially, the oligomerisation domain sofar considered as an inert part of P, plays a crucial role in the replicative machinery
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Le, Bot Ronan Grovel Olivier. "Détection colorimétrique des toxines diarrhéiques par inhibition des protéines phosphatases préparées extemporanément." [S.l.] : [s.n.], 2004. http://theses.univ-nantes.fr/thesemed/PHlebot.pdf.
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Davis, Anthony John. "Characterization of the protein phosphatase 2A regulatory subunit PR70." Access to abstract only; dissertation is embargoed until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=123.
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Freitas, Maria João Martinho de. "Addressing sperm motility regulation through characterization and modulation of the GSK/PPP1R2/PPP1 signaling pathway." Doctoral thesis, Universidade de Aveiro, 2018. http://hdl.handle.net/10773/22772.
Abstract:
Doutoramento em BiologiaSperm motility acquisition and maintenance is a fundamental process for oocyte fertilization and consequently conception. The signaling events underling sperm motility acquisition have been studied for decades. However, many questions are still unanswered. Also, the limited options currently available for male contraception (condom, vasectomy and withdrawal) reflect the necessity of a new group of male contraceptives based on sperm motility modulation. GSK3/PPP1R2/PPP1 signaling pathway is involved in sperm motility acquisition during epididymis transit. The main goal for this work was to deepen the knowledge on the signaling events involved in human sperm motility by focusing on the characterization and modulation of the signaling pathway GSK3/PPP1R2/PPP1. We first designed, synthetized and characterized a disruptive bioportide based on cell penetrating peptide technology. In vitro studies revealed that the disruptive bioportide interferes with PPP1R2/PPP1CC2 interaction and restores PPP1CC2 activity. We also demonstrated that when exposed to the disruptive bioportide, sperm motility is significantly reduced. Aiming to identify sperm protein-protein interactions suitable for pharmacological intervention, we turn our attention to GSK3, a modulator of PPP1R2/PPP1CC2 interactions in sperm. We provide for the first time GSK3 human testis and sperm interactomes. We reported an isoforms specific role for GSK3 in human sperm motility and an in silico analysis revealed GSK3 and GSK3 interactions involved in sperm motility and potential targets for pharmacological intervention. In conclusion, we demonstrated that it is possible to target protein-protein interactions and modulate sperm complexes involved in motility using bioportides. Moreover, we identified new potential protein interactions involved in sperm motility and showed that the development of new type of male contraceptive based on inhibiting sperm motility is now achievable.
A aquisição e manutenção da mobilidade do espermatozoide é fundamental para a fertilização do oócito e consequentemente conceção. Durante décadas, as vias de sinalização necessárias à aquisição de mobilidade por parte do espermatozoide foram alvo de intensos estudos. Contudo, este processo ainda não é inteiramente conhecido. Ademais, as limitadas opções disponíveis para contraceção masculina (preservativo, vasectomia e coito interrompido) refletem a necessidade de desenvolver um contracetivo masculino baseado na modulação da mobilidade do espermatozoide. A via de sinalização GSK3/PPP1R2/PPP1 está envolvida na aquisição de mobilidade do espermatozoide ao longo do transito do epidídimo. O objetivo principal deste trabalho é enriquecer o conhecimento dos eventos celulares necessários na mobilidade do espermatozoide através da caracterização e modulação da via de sinalização GSK3/PPP1R2/PPP1 em espermatozoides humanos. Desenhámos, sintetizámos e caracterizámos um bioportide que quebra interações proteicas baseado em tecnologia de cell penetrating peptides. Estudos in vitro revelaram que o bioportide de ruptura interfere com a interação PPP1R2/PPP1CC2 e é capaz de restabelecer a atividade da PPP1CC2. Também demonstramos que o bioportide reduz significativamente a mobilidade do espermatozoide. Com o objetivo de identificar interacções proteína-proteína adequadas à intervenção farmacológica, focámos a nossa atenção na proteína GSK3, um modulador da interação PPP1R2/PPP1CC2 em espermatozoides. Descrevemos pela primeira vez o interactoma da GSK3 no testículo e espermatozoide humanos e reportamos um papel específico da isoforma GSK3 na mobilidade do espermatozoide. Uma análise in silico revelou interatores da GSK3 e GSK3 que estão envolvidos na mobilidade do espermatozoide e potencialmente poderão ser alvos de intervenção farmacológica para um novo contraceptivo masculino. Em conclusão, demonstramos que é possível provocar a quebra de interações proteína-proteína e modular a mobilidade do espermatozoide usando de bioportides. Também identificamos potenciais novas interações proteicas envolvidas na mobilidade do espermatozoide. Finalmente, mostramos que é possível idealizar um novo tipo de contracepção masculina baseado na inibição da mobilidade do espermatozoide.
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Rochette, Samuel. "Utilisation de pertubations environnementales et génétiques du réseau d'interactions protéine-protéine pour disséquer des processus cellulaires." Master's thesis, Université Laval, 2014. http://hdl.handle.net/20.500.11794/25483.
Abstract:
Les protéines sont les machines moléculaires permettant à la cellule d’accomplir des fonctions biologiques. Pour accomplir ces fonctions, les protéines interagissent souvent entre elles de manière réversible et régulable, ce qui permet à la cellule de s’adapter rapidement à un environnement souvent instable en modulant ces interactions. Par conséquent, l’étude de la dynamique des interactions protéine-protéine est essentielle pour comprendre comment les cellules s’adaptent à diverses perturbations. Les deux chapitres de ce mémoire illustrent respectivement le développement d’une méthode pour identifier et quantifier des modulations d’interactions protéine-protéine en réponse à des perturbations environnementales et comment ces interactions peuvent être utilisées pour disséquer le réseau de régulation d’une protéine phosphatase, la calcineurine, à l’aide de perturbations génétiques. Ensemble, ces deux chapitres illustrent comment l’utilisation d’approches de perturbation du réseau d’interactions protéine-protéine permet de disséquer des processus cellulaires complexes.Proteins are the molecular machines allowing the cell to accomplish a myriad of biological functions. To do so, proteins physically interact with each other in a reversible and tunable way, providing the cell a mechanism to quickly adapt to a changing environment. Thus, studying the dynamics of protein-protein interactions is key in understanding how cells adapt to various perturbations. The chapters included in this thesis illustrate the development of a method to identify and quantify changes in protein-protein interactions in response to environmental perturbations and how protein-protein interactions can be used as reporters to dissect the regulatory network of a protein phosphatase, calcineurin, using a network perturbation approach. Together, these two chapters illustrate the utility of network perturbation approaches to dissect complex cellular processes.
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Höhn, Maximilian [Verfasser], Henning [Akademischer Betreuer] Ebelt, Joachim [Akademischer Betreuer] Neumann, and Peter [Akademischer Betreuer] Boknik. "Auswirkungen der transgenen Überexpression der Phosphoprotein Phosphatase 2A im chronischen Myokardinfarktmodell der Maus / Maximilian Höhn ; Henning Ebelt, Joachim Neumann, Peter Boknik." Halle, 2016. http://d-nb.info/1116955997/34.
46
Polesky, Aaron Joseph Michael. "The Ca²§+/CaM-dependent phosphoprotein phosphatase calcineurin, its role in chemotaxis of axenic cells and analysis of possible substrates in dictyostelium discoideum." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0005/MQ28812.pdf.
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Richard, Charles-Adrien. "La protéine M2-1 du virus respiratoire syncytial : structure et interactions avec des partenaires viraux et cellulaires." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLV029.
Abstract:
Le Virus Respiratoire Syncytial (VRS) est le principal agent responsable d’infections respiratoires sévères chez les nourrissons et les veaux. Le génome du VRS est constitué d’un ARN simple brin de polarité négative qui est répliqué et transcrit par le complexe ARN-polymérase viral (RdRp). Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur anti-terminateur de transcription M2-1. Le but de ce travail était de mieux caractériser la structure et le fonctionnement de deux protéines du complexe RdRp: P et M2-1.M2-1 est un tétramère constitué de 4 domaines : un « doigt de zinc », un domaine d’oligomérisation hélicoïdal, une région flexible, un domaine globulaire interagissant avec l'ARN et P, et une région C-terminale désordonnée. À partir de la structure cristalline de M2-1 pleine longueur, j'ai identifié des résidus critiques sur le doigt de zinc et la région flexible pour l'activité d'anti-terminaison de transcription de M2-1.Par la suite j'ai identifié une région de P critique pour l’interaction P - M2-1 et montre qu’elle est nécessaire au recrutement de M2-1 dans des corps d’inclusion cytoplasmiques. Je montre également que la déphosphorylation de M2-1, nécessaire à la transcription virale, est modulée par un complexe formé entre P et la phosphatase cellulaire PP1.Enfin, la cyclopamine, composé chimique naturel, inhibe la réplication du VRS. Je démontre qu'une seule mutation R151K sur M2-1 est suffisante pour conférer une résistance virale à la cyclopamine. Ces données ouvrent de nouvelles perspectives pour le développement de futures thérapies contre le VRSRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and calves. The RSV genome consists of a single strand, negative-sense RNA, which is replicated and transcribed by the viral RNA-dependent RNA polymerase complex (RdRp). This complex is composed of the nucleoprotein N, the large protein L, the phosphoprotein P and the transcription anti-terminator M2-1. The aim of this work was to better characterize the structure and function of P and M2-1.M2-1 is a tetramer with 4 domains: a zinc-finger, a helical oligomerization domain, a flexible region, a RNA and P binding core domain and a C-terminal disordered region. Based on the crystal structure of the full-length M2-1 protein, I identified residues in the zinc-finger and the flexible loop critical for M2-1 antitermination activity.Then I identified a region of P critical for P – M2-1 interaction and show that it is required for the recruitment of M2-1 to cytoplasmic inclusion bodies. I also show that M2-1 dephosphorylation, which is critical for viral transcription, is modulated by a complex formed by P and the cellular phosphatase protein-1 (PP1).Finally cyclopamine, a natural chemical compound, inhibits the RSV replication. I show that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine. These data open a new avenue for the development of future therapies against RSV infection
48
Guillonneau, Maëva. "Étude de la nucléophosmine NPM dans la réponse de l'endothélium à un stress oxydant majeur." Doctoral thesis, Université Laval, 2016. http://hdl.handle.net/20.500.11794/26990.
Abstract:
Le compartiment microvasculaire est une cible importante du stress oxydant qui est un facteur majeur de la dysfonction endothéliale, notamment au cours d’exposition aux rayonnements ionisants. L’altération de l’endothélium induite par le stress oxydant est impliquée dans la toxicité radio-induite des tissus sains. Limiter les dysfonctions endothéliales est donc un enjeu important des traitements radiothérapeutiques actuels. Cet objectif nécessite une meilleure caractérisation de la signalisation du stress oxydant dans les cellules endothéliales. La voie p38 MAPK est incontournable dans la réponse au stress oxydant mais reste encore insuffisamment caractérisée. Par une approche protéomique, nous avons identifié la nucléophosmine (NPM) comme nouveau partenaire de p38 dans le cytoplasme des cellules endothéliales. La phosphatase PP2a est aussi associée à ce complexe NPM/p38. Nos travaux montrent que le stress oxydant (H2O2, 500μM) régule la déphosphorylation de NPM via PP2a, entraine sa dissociation rapide du complexe et favorise sa translocation vers le noyau. De plus, nous montrons que la présence de NPM déphosphorylée au noyau altère la réponse des cellules aux dommages à l’ADN induits par le stress oxydant. Le céramide sphingolipide membranaire est également un facteur important des voies de stress, particulièrement dans les cellules endothéliales. Notre étude aborde donc l’implication de ce sphingolipide dans la régulation de la voie NPM/p38. Une meilleure caractérisation de la voie p38 et de ses acteurs permettra d’identifier de potentielles cibles afin de limiter les dysfonctions endothéliales et leurs conséquences délétères sur les tissus environnants.The microvascular compartment is a significant target of oxidative stress that is a major factor in endothelial dysfunction, especially during exposure to ionizing radiation. The alteration of endothelium induced by oxidative stress is involved in radiation-induced toxicity of normal tissues. Limiting endothelial dysfunction is therefore an important issue of current radiotherapeutic treatments. This objective requires a better characterization of oxidative stress signaling in endothelial cells. p38 MAPK pathway is essential in oxidative stress response but still insufficiently characterized. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in the cytoplasm of endothelial cells. PP2a phosphatase is also associated with the NPM/p38 complex. Our work shows that oxidative stress (H2O2, 500μM) regulates the NPM dephosphorylation via PP2a, causes a rapid dissociation of the complex, and promotes its translocation to the nucleus. In addition, we show that the presence of NPM dephosphorylated at T199 in the nucleus alters the cellular response to DNA damage induced by oxidative stress. The membrane sphingolipid ceramide is also an important factor in stress pathways, particularly in endothelial cells. Our study describes the involvement of this sphingolipid in the regulation of NPM/p38 pathway. A better characterization of the p38 pathway and its actors provided by our study will identify potential targets in order to limit endothelial dysfunction and its deleterious effect on surrounding tissues.
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DESDOUITS, FREDERIC. "La darpp-32 (dopamine- and camp-regulated phosphoprotein mr=32,000) : un inhibiteur de la proteine phosphatase 1 regule par la phosphorylation de multiples residus." Paris 6, 1995. http://www.theses.fr/1995PA066303.
Abstract:
La darpp-32 (dopamine- and camp-regulated phosphoprotein, mr=32,000) est une proteine inhibitrice de la proteine phosphatase 1 (pp 1) dont l'activite est regulee par la phosphorylation de plusieurs residus. La darpp-32 devient un puissant inhibiteur de la pp 1 lorsqu'elle est phosphorylee sur la threonine-34 par la kinase activee par l'ampc (pka). La darpp-32 est aussi un substrat de la caseine kinase ii. Pour permettre les etudes biochimiques et physico-chimiques, la darpp-32 de rat a ete exprimee dans la bacterie et purifiee en grande quantite. Les etudes conformationnelles nous ont indique que la darpp-32 comprend un domaine amino-terminal relativement rigide et un domaine carboxy-terminal plus flexible. La phosphorylation de la threonine-34 ne modifie pas la conformation globale de la darpp-32, mais cree un site de relativement faible affinite pour la proteine phosphatase 1 qui, en se combinant avec un second site de faible affinite non-regule par phosphorylation, transforme la darpp-32 en un inhibiteur de haute affinite pour la phosphatase. La phosphorylation de la darpp-32 par la caseine kinase ii n'induit qu'une faible modification de la structure de la darpp-32, intervenant essentiellement dans la partie rigide carboxy-terminale de la proteine. Nous avons ensuite mis en evidence la phosphorylation de la darpp-32 sur la serine-137 par la caseine kinase i dans les neurones striatonigraux et etudie les parametres biochimiques de cette reaction in vitro. La phosphorylation de la serine-137 accelere, de facon inhabituelle, la migration de la darpp-32 en gel de polyacrylamide en presence de sds. La phosphorylation de la darpp-32 par la caseine kinase i inhibe la dephosphorylation de la threonine-34 par la calcineurine, in vitro et dans les neurones striatonigraux
50
Fernandes, Emanuel Ferreira. "The role of Synphilin-1A/PPP1 complex in Lewy bodies formation." Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12488.
Abstract:
Mestrado em Biomedicina MolecularOne of the major Parkinson’s disease hallmarks is the development of cytoplasmic inclusions, termed Lewy bodies, mainly within surviving neurons in the brainstem of affected patients. Many proteins have been identified in the Lewy bodies, but their formation mechanism remains unclear. Among the proteins already identified in the Lewy bodies are synphilin-1, a α-synucleininteracting protein, and synphilin-1A, a synphilin-1 splice variant. Synphilin-1 and synphilin-1A have been considered key elements in Parkinson’s disease as their overexpression in human embryonic kidney 293 cells, with or without α-synuclein, leads to the formation of Lewy body-like cytoplasmic inclusions. Therefore, efforts have been made to clarify the regulatory mechanisms behind synphilin-1 and synphilin-1A aggregation as a means to uncover new aspects of Lewy bodies formation. Although kinases able to phosphorylate synphilin-1 have been described, there are no specific data concerning the phosphatases responsible for its dephosphorylation. This gap was filled with the identification of synphilin-1A as a novel phosphoprotein phosphatase 1-interacting protein in human brain, through yeast two hybrid. Hence, in the present work, the physiological role of synphilin-1A/phosphoprotein phosphatase 1 complex is studied, being demonstrated the ability of synphilin-1A to specifically target phosphoprotein phosphatase 1 to inclusion bodies, using immunofluorescence experiments. Moreover, the consequences of disrupting this interaction are explored using a synphilin-1A mutant unable to interact with phosphoprotein phosphatase 1, revealing an enhancement of synphilin-1A aggregative properties. Also, the ability of wild type synphilin-1A and the mutant form to produce aggresomes upon overexpression and without proteasome inhibition is addressed but the results are unclear, does not allowing the classification of the inclusions documented in this work as aggresomes. All together, these results suggest that synphilin-1A is able to affect phosphoprotein phosphatase 1 targeting within cells, being inclusion bodies formation dependent and, most specifically, controlled by phosphoprotein phosphatase 1 activity. It is postulated that decreased phosphoprotein phosphatase 1 recruitment to inclusion bodies produces hyperphosphorylated states that favor protein aggregation.
Uma das características principais da doença de Parkinson é o aparecimento de inclusões citoplasmáticas, chamadas corpos de Lewy, maioritariamente nos neurónios dopaminérgicos remanescentes, no tronco cerebral dos pacientes afetados. Várias proteínas têm sido identificadas nos corpos de Lewy, mas o seu mecanismo de formação permanece por clarificar. Entre as várias proteínas já identificadas encontram-se a sinfilina-1, uma proteína interactora da α-sinucleina, e a sinfilina-1A, uma variante da sinfilina-1. Ambas têm sido consideradas elementos chave na doença de Parkinson, já que a sua sobreexpressão em células embrionárias 293 de rim humano, com ou sem a α-sinucleina, conduz à formação de inclusões citoplasmáticas parecidas com corpos de Lewy. Posto isto, têm sido envidados esforços no sentido de clarificar os mecanismos reguladores da agregação da sinfilina-1 e da sinfilina- 1A, como forma de revelar novos aspetos da formação dos corpos de Lewy. Embora tenham sido descritas cinases capazes de fosforilar a sinfilina-1, não há informações concretas sobre as fosfatases responsáveis pela sua desfosforilação. Este vazio começou a ser preenchido com a identificação da sinfilina-1A como uma nova proteína interactora da fosfoproteína fosfatase 1 em cérebro humano, através do sistema dois híbrido de levedura. Deste modo, no presente trabalho, procede-se ao estudo da função fisiológica do complexo sinfilina-1A/fosfoproteína fosfatase 1, demonstrando-se a capacidade da sinfilina-1A de recrutar de forma específica a fosfoproteína fosfatase 1 para corpos de inclusão, com recurso a imunofluorescência. Adicionalmente, as consequências do bloqueio desta interação são exploradas utilizando um mutante da sinfilina-1A incapaz de interagir com a fosfoproteína fosfatase 1, revelando um aumento das propriedades agregativas da sinfilina-1A. Finalmente, também é avaliada a capacidade de a sinfilina-1A selvagem e mutada produzirem agressomas, quando sobreexpressas e sem inibição do proteassoma, mas os resultados não são claros e não permitem a classificação das inclusões documentadas no presente trabalho como agressomas. Em conjunto, estes resultados sugerem que a sinfilina-1A tem a capacidade de afetar o endereçamento da fosfoproteína fosfatase 1 nas células, sendo a formação de corpos de inclusão dependente e, mais concretamente, controlada pela atividade da fosfoproteína fosfatase 1. Postulase que um menor endereçamento da fosfoproteína fosfatase 1 para os corpos de inclusão conduza a estados hiperfosforilados que favorecem a agregação proteica.