Dissertations / Theses on the topic 'Immunophilins'
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1
Dornan, Jacqueline. "Immunophilins : an investigation into function and structure." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/22159.
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The work described in this thesis focused on the over-expression, purification, biochemical characterisation, crystallisation and 3-D structure determination of three members of the immunophilin family. The overall goal of the project was to use biochemical and structural information derived from these studies to assist in the elucidation of the various roles played by these proteins in diverse systems. Cycophilin 3. Cloning and expression of eleven cyclophilin homologues from the free-living nematode Caenorhabditis elegans (C. elegans) has recently been reported. Cyclophilin 3 (Cyc-3), one of the most abundantly expressed isoforms, was chosen as the initial target for further study. The structure of the protein was solved to high resolution, allowing identification of a number of key structural features that differ from those of the archetypal cyclophilin, Cyclophilin A. These newly determined features, which may prove to be functionally significant, were used to define a new cyclophilin subfamily. Cycophilin 40. Cyclophilin 40 (Cyp 40) is a two domain immunophilin containing a conserved cyclophilin domain linked via a charged linker region to the C-terminal domain comprised largely of three copies of the tetratricopeptide repeat (TPR) motif. Cyp 40 constitutes part of the “mature” steroid hormone receptor complex, interactions with Hsp90 have been described, although the exact nature of the interaction has not been well defined. Using cloned bovine Cyp 40, two very different crystal forms were grown. FKBP22. Isolation of FKBP22 for the endoplasmic reticulum of Neurosporra crassa (N. crassa) has recently been reported. The C-terminal sequence appears to be unique among other FKBP sequences, being rather highly charged and is consistent with an amphipathic helical conformation. Crystallisation and preliminary partial X-ray structure determination of FKBP22 from N. crassa are reported.
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Morgan, Gloria Yvonne. "The expression of immunophilins in cells and organelles." Thesis, University of Sussex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282145.
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McKenzie, Neil Iain. "The immunophilins as drug targets : development of novel fluorescence assays." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17961.
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The immunophilins are a superfamily of proteins comprising the cyclophilins, the FKBPs and the parvulin sub-families. Members are present ubiquitously in plant and animal cells, acting as both prolyl-isomerases and signalling proteins. Some also have chaperone activity. The prolyl isomerase function of the immunophilins has been identified as being central to progression of a large number of diseases, making them tempting drug targets. Whilst there are several assays which can be used to identify inhibitors of the prolyl isomerase function, they are hampered by one or more problems: multistep mechanisms, poor signal-to-noise ratios, expensive, laborious and unamenable to high throughput screening. Multiple fluorescent systems (fluorescence anisotropy, FRET, 2D-FIDA/FCS) and several technologies (solution and solid phase synthesis, solution and solid phase screening, combinatorial synthesis, and stopped-flow spectrometry) were explored to develop a system suitable for fast, efficient screening of immunophilins. The most promising of these is a prototype assay based on the design, cloning, expression and production of fluorescently labelled mutant of cyclophilin B, which shows an increase in fluorescence emission upon cyclosporin ligand binding.
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McCann, Fiona Elizabeth. "Studies on novel immunophilins and the effects of immunosuppressant drugs on neurons." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246593.
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Davies, Todd Howard. "Regulation of glucocorticoid receptor function by associated TPR-domain proteins." Connect to full-text via OhioLINK ETD Center, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1098292002.
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Thesis (Ph. D.)--Medical College of Ohio, 2003."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin Sanchez. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 100-124).
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Davies, Todd Howard. "Regulation of Glucocorticoid Receptor Function by TPR-domain Proteins." University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1098292002.
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Cluning, Carmel. "Steroid receptor-associated immunophilins : influence of targeted knockdown and altered expression on receptor signalling." University of Western Australia. School of Medicine and Pharmacology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0215.
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[Truncated abstract] Steroid receptors belong to the superfamily of nuclear receptors, and include the androgen receptor (AR), estrogen receptors (ER[alpha] and ER[beta], glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the progesterone receptors (PRA and PRB). Before binding ligand, the receptor undergoes biochemical and structural modifications through a series of interactions with molecular chaperones and cochaperones all within a receptor heterocomplex. The mature receptor complexes with the major chaperone Hsp90, the stabilising cochaperone p23, and one member of a group of cochaperones termed immunophilins. Steroid receptor-associated immunophilins include the cyclophilin, CyP40, two FK506-binding proteins, FKBP51 and FKBP52, and the protein phosphatase, PP5. Immunophilins are characterised by the presence of TPR domains which compete directly for the TPR-acceptor site within Hsp90. This leads to mutually exclusive, immunophilin-containing receptor complexes. While PP5 contains a C-terminal phosphatase domain, CyP40, FKBP51 and FKBP52 each contain an N-terminal peptidyl prolyl isomerase (PPIase) domain, which catalyses the cis/trans isomerisation of prolyl peptide bonds. FKBP52 has been demonstrated to potentiate the ligand-dependent activity of AR, GR and PR, but not ER[alpha]. Knowing that CyP40 is the preferred immunophilin associated with the ER[alpha] heterocomplex, it was hypothesised that this immunophilin plays a role in ER[alpha] function. ... As all mutants maintained this potentiating activity it was concluded that the five altered residues found within gpGR do not contribute to the altered interaction of FKBP52 and receptor. However, it cannot be discounted that FKBP51 is more competitive for gpGR. Immunophilins are hormonally regulated, with FKBP52 found to be essential for female fertility in mice. It was hypothesised that levels of immunophilins, associated with steroid receptors important in the menstrual cycle, would be regulated to reflect hormonal activity within cycling endometrium. Human pre-menopausal endometrial sections taken from different phases of the menstrual cycle were examined immunohistochemically for expression of CyP40, FKBP51, FKBP51 and PP5. Immunophilin levels peaked at the mid-secretory phase correlating with stromal decidualization, a process essential for eventual blastocyst implantation. The importance of immunophilins to steroid receptor action was therefore reinforced by the observation that immunophilins appear to be hormonally regulated in cycling pre-menopausal human endometrium. Further studies into the effects of immunophilin loss and knockdown on steroid receptor-mediated responses in specific mouse tissues, knockout-derived mouse embryo fibroblasts and cancer cell lines may contribute to our understanding of the receptor-selective and tissue-specific actions of the immunophilins. Elucidation of the mechanisms through which they modulate receptor function may provide opportunities for therapeutic intervention in steroid-related disease.
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Warrier, Manya. "Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1223923687.
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Sandhu, Khushwant Singh. "Identification and molecular characterization of the putative immunophilins (IMMs) in the oilseed rape pathogens Leptosphaeria maculans, Leptosphaeria biglobosa, and Plasmodiophora brassicae." Doctoral thesis, Česká zemědělská univerzita v Praze, 2016. http://www.nusl.cz/ntk/nusl-259691.
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Oilseed rape is largely infected by several phytopathogens and two most economical important diseases are blackleg caused by fungus species complex Leptosphaeria maculans and L. biglobosa and clubroot caused by protist P. brassicae. The sequenced genomes of these phytopathogens provide opportunity to uncover various aspects related to disease infection, host pathogen interactions, plant disease resistance, and evolution of pathogens. Considering these we focused on one of the most conserved family called immunophilins (IMMs) in these genomes. IMMs are comprised of three structurally unrelated sub-families including cyclophilins (CYPs), FK506-binding proteins (FKBPs), and parvulin-like proteins (PARs). We identified putative members of IMMs in each phytopathogen using bioinformatics approaches. We further characterized the IMMs based on domain architecture, subcellular localization, exon-intron organization, transcriptomic expression patterns, gene ontology terms, conserved motifs presents and evolutionary analyses. IMMs are performing several vital roles in plants, animals and fungi. However, in phytopathogens their roles are not well established except for cyclophilin that implicates in pathogenicity in some phytopathogens. Therefore, we exploited the role of cyclophilin in L. maculans and L. biglobosa using expression profiles and in P. brassicae using Magnaporthe oryzae cyclophilin deletion mutant. Overall, we concluded that the cyclophilin acts as a virulence determinant in our studied phytopathogens. However, delineating the precise role of other IMMs would also be imperative. Taken together, our findings for the first time shed light on the highly conserved IMM family in the oilseed rape pathogens.
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Somarelli, Jason Andrew. "The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation." FIU Digital Commons, 2009. http://digitalcommons.fiu.edu/etd/83.
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Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.
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Kontopidis, George A. "Immunophilin ligand design." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/22386.
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The general aim of the project is predict and select small molecule ligands which may bind to 3D protein templates. Proteins from the Immunophilin family were used. The putative ligands were selected by two different methods, by structure similarity and using the docking program LIDAEUS, which was developed in house by Dr. P. Taylor. Twenty nine small molecules were selected from a small molecule database and were tested with a fluorescence assay, a PPIase assay and x-ray crystallography for binding activity. Six new ligands have been discovered which bind to Cyp-A. In this work the IC50 (concentration in which half inhibition occurred) of the putative ligands were determined by rotamase inhibition assay. In addition the binding of the ligands was also confirmed by crystallographic experiments. The X-ray structures of 3-acetyl-1 methyl piperidine, ethyl-piperidine glyoxylate, dimethyl sulfoxide, tetramethylene sulfoxide, cyclopentanone and 5,5-dimethyl-1,3-cyclohexanedione bound to Cyp-A have been solved and refined with Rfactor for each structure less than 20%. The structural analysis of the native and ligand structures revealed a hydrophobic pocket surrounded by residues Asn 102, Met 61, Arg 55 and His 122, a hydrogen bond donor site of the main chain nitrogen Asn 102, and another hydrogen bond donor site of the guanidinium of Arg 55. The six novel ligands have been classified into 3 different families of compounds, 3-Acetyl-1 methyl piperidine and ethyl-piperidine glyoxylate belong to the piperidine family and share structural similarities with a natural substrate of Cyp-A the dipeptide Ala-Pro. Dimethyl sulfoxide (DMSO), tetramethylene sulfoxide and cyclopentanone form the DMSO family and they do not share any similarities with known Cyp-A ligand. Only one compound has been discovered in the third family. This compound 5,5-dimethyl-1,3-cyclohexanedione also known as dimedone shows some structural similarities with the Val 11 residue of the Cyp-A ligand cyclosporin.
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Yuande, Y. "Structural studies of immunophilin-ligand complexes." Thesis, University of Edinburgh, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.664185.
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The work described in this thesis used crystallographic and biochemical methods to study the complex of immunophins (humans Cyclophilin A (hCypA) and FK506 binding protein (FKBP)) with a series of chemically synthesised ligands. HCypA is the cytosolic receptor of the immunosuppressant Cyclosporin A (CsA), and FKBP is the receptor for the immunosuppressant FK506. Both hCypA and FKBP possess peptidyl-prolyl isomerization (PPIase) enzymatic activity. In this project hCypA was produced and characterised in house. SDS page electrophoresis, mass spectrometry and PPIase enzymatic assays were used to characterise hCypA, which was expressed in E. coli and purified in house with chromatographic techniques. PPIase assays and fluorescence spectrometry were used to identify nine novel ligand complexes. The ligands were synthesised in the Chemistry department of Edinburgh University. Crystal complexes of the nine hCypA ligand complexes were obtained and crystal structures were solved up to a resolution of 1.8 A. It was observed that all nine ligands bind hCypA in a similar mode, and the relationship between the binding strength and structural features was evaluated. The following equation was developed to relate measured dissociation constant (Kd) and structural features: Kd = 238 - 0.185 * (Buried area) = 17.9* (Number of Hydrogen bonds less than 3A) - 6.7 * (Number of Hydrogen bonds greater than 3.0 A) FKBP was also crystallised. The complex crystal of FKBP and the dipeptide leucylproline (Leu-Pro) was prepared. The crystal structure of FKBP and Leu-Pro shows that the amide bond of bound Leu-Pro is twisted and provides an insight into the PPIase mechanism.
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Patterson, Alan F. "Biophysical and crystallographic studies of immunophilin-ligand complexes." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/15599.
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The work in this thesis describes the cloning, expression purification and characterisation of human CypA and FKBP12 proteins. Various biophysical techniques were used to study and characterise the interactions of different ligands with these target proteins. Vectors containing the genes encoding each of these, for expression of both wild-type protein and protein with a hexa-histidine tag, have been cloned for both hCypA and hFKBP12. Crystals of hCypA have been grown, and the structures, in complex with a range of small molecules including dipeptides, and dimedone derived ligands, have been solved. hCypA crystal complexes were also used to further explore the nature of the ‘crystal dissociation constant, Kcryst’ using the depetide ligand Val-Pro. Crystals were soaked in various ligand concentrations and by solving the X-ray structures of these complexes, ligand occupancies could be determined providing an experimental value for the equilibrium binding constant, Kcryst = 23mM. This compares well with a Ki value 19mM measured using an enzyme inhibition assay. Protein-ligand binding data measured using a range of methods is also presented, including fluorescence spectroscopy, mass spectrometry and surface plasmon resonance. It has also been demonstrated how the technique of surface plasmon resonance may be optimised to provide a sensitive ligand-binding assay. By tethering protein to a Ni2+ chip via an N-terminal hexa-his tag followed by covalent linking via primary amines, a stable and active surface was obtained. This allowed a competition binding assay to be developed, which allows the binding of ligands of Mr 250-500Da to be measured. The work done here should help further the design of potential inhibitors of hCypA, and in the measurement of their binding affinities; and possibly aid in the future design of anti HIV-1 therapies.
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Lane-Guermonprez, Lydie. "Stress nitro-oxydatif et immunophilines dans le contrôle du métabolisme cholinergique." Paris 11, 2002. http://www.theses.fr/2002PA112112.
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Le monoxyde d'azote est un modulateur physiologique de l'activité nerveuse qui peut se combiner avec l'ion superoxyde pour former du peroxynitrite, un radical oxydant et nitrant. La calcineurine est la seule phosphatase enrichie dans les neurones, modulée par le calcium et perturbée par certains stress oxydatifs. Elle est impliquée dans les phénomènes de plasticité synaptique et de régénération axonale. Les principaux inhibiteurs de cette phosphatase sont des immunosuppresseurs, le FK506 et la cyclosporine (CsA). Ces agents ont des effets paradoxaux in vivo, puisqu'ils sont à la fois neurotoxiques et neurotrophiques. Pour agir, ces deux composés doivent au préalable se fixer à des enzymes intracellulaires, les immunophilines. Ce sont des chaperones ubiquitaires qui agissent sur l'adressage, l'oligomérisation et l'activité de certaines protéines en modifiant la conformation des résidus prolines qu'elles contiennent. Malheureusement, il est relativement peu aisé de dissocier les effets des immunosuppresseurs qui passent par une inhibition de la calcineurine de ceux qui impliquent les immunophilines. L'objectif de cette thèse était d'évaluer, sur un modèle simple, les effets potentiels du peroxynitrite et des immunosuppresseurs sur le métabolisme cholinergique. Nous avons utilisé deux approches complémentaires sur des synaptosomes de l'organe électrique de Torpille. L'étude des effets à court terme du peroxynitrite et des immunosuppresseurs sur la synthèse et la libération de l'ACh nous a permis d'identifier la ChAT comme cible du peroxynitrite, et le transporteur de choline à haute affinité comme cible de la calcineurine. Dans une deuxième approche, nous avons montré que la synapsine est un partenaire possible de la cyclophiline B, et que leur interaction est abolie par la CsA. Cela suggère un rôle pour la cyclophiline B dans la régulation du stockage et de la libération des neuromédiateursNitric oxide is a physiological modulator of neuronal functions, which can combine with superoxide to form peroxynitrite. Peroxynitrite is a powerful, short-lived oxidizing and nitrating agent. Calcineurin is the only known calcium-dependent phosphatase. It is enriched in neurons and particularly sensitive to oxidative stress. It plays important roles in synaptic plasticity and axonal regeneration. Available inhibitors of calcineurin are the immunosuppressors cyclosporin A (CsA) and FK506. When clinically administered, these drugs display bath neurotoxic and neurotrophic effects, that are not fully understood. To be able to inhibit calcineurin activity, they first have to complex with intracellular enzymes named immunophilins. Immunophilins are ubiquitously expressed chaperones endowed with cis-trans prolyl isomerase activity. By modifying praline conformation, they can regulate the function, export and oligomerization of numerous proteins, notably transporters and channels. Unfortunately, it is quite uneasy to separate the effects of immunosuppressors linked to calcineurin inhibition from their effects on immunophilins. The goal of this work was to determine, on a simple model of nerve endings, whether peroxynitrite or immunosuppressors could modify acetylcholine metabolism. These questions were assessed by two complementary approaches on the same model, ie purely cholinergic synaptosomes isolated from Torpedo electric organ. First, we studied the short-term effects of peroxynitrite and immunosuppressors on synaptosomal ACh synthesis and/or release. We identified ChAT as a major target of peroxynitrite and the high affinity choline transporter as a target of calcineurin. In a second approach, we searched for cyclophilin ligands in synaptosomes, and found that synapsin can interact with cyclophilin B in a CsA and calcium dependent way. This suggests an important role of cyclophilin B in mid- or long-term regulation of neuromediator storage and release
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Guimiot, Fabien. "Etude fonctionnelle de deux immunophilines, Fkbp25 et Fkbp36 dans le développement neuronal." Paris 7, 2003. http://www.theses.fr/2003PA077217.
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Chopard, Christophe. "Régulations de la sécrétion et de l’activité biologique de la protéine Tat du VIH-1 : rôles de la palmitoylation et de Gag." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20089/document.
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La protéine du VIH-1 est une protéine essentielle à la transcription et à la réplication du virus. Elle a donc un rôle crucial dans la cellule infectée. On sait qu'une partie de la Tat cellulaire peut être sécrétée, malgré l'absence de séquence signal. En effet, les 2/3 de la Tat cellulaire sont exportés par la cellule T-primaire infectée. Le mécanisme de sécrétion de Tat est non conventionnel et se produit directement à travers la membrane plasmique où Tat est recrutée grâce à sa très forte affinité pour le phosphatidylinositol(4,5)-biphosphate ou PI(4,5)P2 qui est exclusivement localisé à ce niveau. La Tat extracellulaire a un effet délétère sur de nombreux types cellulaires et agit donc comme une toxine virale. Elle constitue un facteur déterminant de l'évolution vers le SIDA. En accord avec cette efficacité de sécrétion par les cellules T primaires infectées, Tat est essentiellement localisée sur la membrane plasmique des T primaires infectées par le VIH-1. Une fraction importante de Tat est donc résidente à la membrane et nous avons recherché un mécanisme pouvant expliquer cette rétention et mis en évidence la palmitoylation. Nos travaux montrent que Tat est palmitoylée, dans des cellules T ainsi que dans les ‘cibles' comme les neurones et les macrophages. Cette palmitoylation, qui inhibe la sécrétion de Tat, est réalisée sur la cystéine 31 de Tat (qui possède 7 cystéines) par l'enzyme DHHC20 avec comme cofacteurs nécessaires les immunophilines (prolyl-isomérases), Cyclophiline A et FKBP12, qui interagissent avec Tat au niveau de la membrane. Nos résultats indiquent aussi qu'en présence de Gag, la palmitoylation de Tat est inhibée. Nous pensons que l'export de la CypA dû à son encapsidation diminuerait la quantité de CypA disponible pour Tat, inhibant la palmitoylation de Tat et permettant sa sécrétion efficace par les cellules infectées. En effet, le VIH-1 encapside 250 copies de CypA/virion, le taux de CypA régulant la virulence des virions produits. Dans les cellules cibles, Tat serait fortement palmitoylée ce qui induirait sa fixation quasi irréversible au PI(4,5)P2, l'empêchant de ressortir de la cellule et permet ainsi un effet cumulatif des doses reçues par la cellules. Cette accumulation de Tat perturbe des processus membranaires dépendant du PI(4,5)P2 tels que la phagocytose et la neurosécrétion. La palmitoylation de Tat est nécessaire pour ces effets. Ces actions de la Tat extracellulaire pourraient participer au développement des infections opportunistes et des troubles neurologiques observé lors du SIDAHIV-1 Tat is a small protein that is required for viral transcription and multiplication. It thus has a crucial role in the infected cell. It was known that Tat can be secreted despite its lack of signal sequence. In fact 2/3 of cellular Tat are exported by infected primary T-cells. The unconventional secretion of Tat relies on its interaction with phosphatidylinositol(4,5)-biphosphate or PI(4,5)P2, a lipid that is concentrated within the inner leaflet of the plasma membrane and is strictly required for Tat secretion. Exogenous Tat has deleterious effects on several cell types, indicating that extracellular Tat is involved in evolution to AIDS. Consistent with this secretion efficiency, Tat is mainly localized at the plasma membrane of primary T-cells infected by HIV-1. A large fraction of Tat is resident at the membrane and we looked for a mechanism that could explain this retention and discovered that Tat is palmitoylated. Our studies show that Tat is palmitoylated, both in T-cells and also in ‘target' cells such as neurons and macrophages. Tat palmitoylation inhibits its secretion and is performed on Tat cysteine 31 (Tat has seven cyteines) by the enzyme DHHC20 using immunophilins (prolyl ismerases), Cyclophilin A (CypA) and FKPB12, as cofactors. Our results also indicate that the presence of Gag inhibits Tat palmitoylation. We believe that the export of CypA due to its encapsidation will make less CypA available for Tat, thereby inhibiting Tat palmitoylation. Indeed, HIV-1 encapsidates 250 copies of CypA/virion and the amount of CypA regulates the virulence of produced virions. In target cells, Tat is strongly palmitoylated and this modification induces its almost irreversible binding to PI(4,5)P2, preventing its secretion and allowing cumulative effect of minute Tat doses.Tat palmitoylation enables Tat to severely inhibit various PI(4,5)P2-dependent processes such as phagocytosis and neurosecretion. These effects of extracellular Tat likely contribute to the development of opportunistic infections and neurological disorders observed during AIDS
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Rivière, Sylvie. "Caracterisations biochimiques et fonctionnelles d'une immunophiline, la fkbp25, et d'une cytokine, le mif." Paris 11, 1995. http://www.theses.fr/1995PA112133.
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Cette these est consacree a l'etude des fonctions de deux proteines: la fkbp25, qui est une immunophiline, et le mif, une cytokine. Les immunophilines sont les proteines acceptrices, intracellulaires pour la plupart d'entre elles, des immunosuppresseurs que sont la cyclosporine a, le fk506 et la rapamycine. Ces derniers empechent l'activation du lymphocyte t, consecutive au contact avec un immunogene, et bloquent ainsi la reponse immunitaire. Nous avons, dans un premier temps, purifie la fkbp25 bovine a partir d'organes et obtenu un antiserum de lapin. Nous avons ainsi montre apres analyse par une technique d'immunotransfert de fractions cellulaires, que la fkbp25 est, du moins dans le lymphocyte t, une proteine nucleaire. De plus, elle lie in vitro l'adn contrairement a la fkbp12, une autre immunophiline, de sequence en acides amines proche. La comparaison des sequences de ces deux proteines suggere que le site potentiel de liaison a l'adn se trouve localise dans le domaine n-terminal de la fkbp25. Nous avons de plus identifie dans cette region un site potentiel de liaison a l'adn de type helix-loop-helix. Il est donc possible que la fkbp25 soit impliquee dans les mecanismes nucleaires conduisant a la division et a la proliferation de la cellule. La deuxieme proteine que nous avons etudiee est le mif (macrophage migration inhibitory factor). Elle est la premiere cytokine decrite et inhibe in vitro la migration des macrophages a l'exterieur de tubes capillaires. Nous avons montre, grace a un serum polyclonal de lapin anti-mif bovin, que le mif est present dans les lymphocytes t et b, dans les macrophages et dans les fibroblastes. Il est aussi distribue dans tous les organes que nous avons etudies, c'est-a-dire le cur, le cerveau, le poumon, la rate, le rein et le foie. Aucune specificite pour une cellule ou un organe du systeme immunitaire n'a ete mise en evidence. Le mif semble donc etre et ce, contrairement aux cytokines, une proteine constitutive et ubiquiste dans les cellules et organes de mammiferes. Enfin, dans les lymphocytes t, nous avons etabli qu'il existe au moins deux isoformes de la proteine mif. Le mif est decrit comme etant secrete par le lymphocyte t ou le macrophage active. Pourtant, dans nos tests, nous n'avons pas detecte de secretion de mif par trois lignees de lymphocytes t, de macrophages humain et murin. Nous avons aussi montre que le mif n'induit pas la synthese de molecules de classe ii du cmh chez le macrophage, contrairement a ce qui avait ete decrit dans la litterature. Le mif bovin natif et le mifr humain n'ont pas d'activite d'inhibition de la migration des macrophages. Nous rejoignons en cela les travaux de plusieurs autres equipes. Enfin, le mif bovin natif n'a pas l'activite glutathion-s-transferase decrite pour le mif de rat, et ne lie pas le glutathion. Nous avons aussi montre qu'il existe dans le serum, au moins une proteine de liaison du mif, dont la sequence n-terminale est identique a celle de l'albumine. Nous pensons que cette proteine est la sarcolectine. Ces travaux, associes a ceux d'autres equipes, montrent, d'une part, que le mif n'est probablement pas une cytokine, et d'autre part, que son activite d'inhibition de la migration des macrophages est largement contestable
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Le, Bihan Stéphane. "Role des immunophilines dans le mecanisme d'action des recepteurs des glucocorticosteroides et des progestagenes." Paris 11, 1996. http://www.theses.fr/1996PA11T025.
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Guimiot-Maloum, Ismahane. "Analyse fonctionnelle de deux gènes nouveaux, C61 et FKbp25, identifiés par comparaison de transcriptomes : leur implications dans le développement cortical." Paris 6, 2005. http://www.theses.fr/2005PA066309.
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Kamah, Amina. "Identification et caractérisation des modifications post-traductionnelles de la protéine TAU : implication dans le processus d'agrégation." Thesis, Lille 1, 2015. http://www.theses.fr/2015LIL10049.
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Les démences séniles sont caractérisées, au niveau moléculaire, par l’agrégation de quelquesprotéines-clés. On peut donner comme exemple l’α-synucléine, dans la maladie de Parkinson,ou le peptide β-amyloïde et la protéine Tau, dans la maladie d’Alzheimer. Le mauvaisrepliement de ces protéines est souvent évoqué, même si elles n’adoptent pas une structurestable lorsqu’elles sont isolées en solution. La protéine Tau est une protéine neuronaleappartenant à la famille des protéines MAP (protéines associées aux microtubules). Saprincipale fonction est de stimuler la polymérisation de la tubuline en microtubules, assurantainsi le transport cytoplasmique au sein d’une cellule. Tau ainsi que des mutants de Tau ont étéimpliqués dans de nombreuses pathologies neurodégénératives regroupées sous le terme detauopathies, dont la plus connue est la maladie d’Alzheimer. Bien qu’aucune mutation de Taun’ait été décrite dans la maladie d’Alzheimer, Tau joue un rôle-clé dans cette pathologie. Elleest caractérisée par la présence d’agrégats fibrillaires de protéines Tau hyperphosphoryléesnommés PHF (Paired Helical Filaments) qui envahissent progressivement le cerveau. Cesobservations sont associées à un déséquilibre entre phosphorylation et déphosphorylation.Malgré les différentes études menées sur le sujet, le mécanisme d’agrégation n’est pas compriset plusieurs pistes sont envisagées, notamment l’implication des modifications posttraductionnelles.Nous avons déterminé le profil de modifications de la protéine Tau parspectroscopie RMN et étudier leur implication dans le processus d’agrégation parspectrophotométrie. Parmi les modifications covalentes existantes, nous avons étudiél’acétylation des résidus lysine et l'O-β-N-acétylglucosaminylation des résidus sérine etthréonine. La troisième est une modification conformationnelle qui consiste à stimulerl’échange conformationnel des prolines par des enzymes à activité peptidyl prolyl cis-transisomérase (PPIase)Senile dementia are characterized by protein aggregation such as α-synuclein in Parkinsondisease or β-amyloid peptide and Tau protein in Alzheimer disease. Protein misfolding has beenevoked in the pathological processes even though these proteins do not adopt a stable threedimensionalstructure in solution. Tau protein belongs to the MAP (Microtubule-AssociatedProtein) family. It stimulates tubulin polymerization into microtubule allowing for cytoplasmictransport in cell. Tau and its mutated forms are found in neurodegenerative diseases,collectively referred to as tauopathy, the most famous being Alzheimer’s disease. Thesepathologies are characterized by fibrillary aggregates of hyperphosphorylated Tau namedPaired Helical Filaments (PHF) that invades gradually throughout the brain. These observationsare correlated with imbalance between phosphorylation and dephosphorylation in AD brains.Despites extensive studies, the aggregation mechanism is still not understood and severalpathways were considered, especially posttranslational modifications other thanphosphorylation. We have investigated the modifications of Tau protein by NMR spectroscopyand studied effect of these modifications on aggregation process by spectrophotometry. Amongcovalent modifications, we have studied lysine acetylation and Ser/Thr O-β-Nacétylglucosaminylation.The third investigated modification is the conformational switch ofproline residues catalyzed by a peptidyl prolyl cis trans isomerase
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Blackburn, Elizabeth Anne. "Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/6504.
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The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors.
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Moore, Stephen. "Characterisation of a novel immunophilin-like gene, repressed by low doses of ionising radiation; identification of interacting proteins." Thesis, University of Ulster, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272344.
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Strunz, Patrick-Pascal Holger [Verfasser], Petra [Gutachter] Eder-Negrin, Erhard [Gutachter] Wischmeyer, and Brenda [Gutachter] Gerull. "Interaktion von TRPC-Ionenkanälen mit dem Immunophilin FKBP52 / Patrick-Pascal Holger Strunz ; Gutachter: Petra Eder-Negrin, Erhard Wischmeyer, Brenda Gerull." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1210862336/34.
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Edvardsson, Anna. "Peptidyl-prolyl cis-trans Isomerases in the Chloroplast Thylakoid Lumen." Doctoral thesis, Linköping : Univ, 2007. http://www.bibl.liu.se/liupubl/disp/disp2007/med983s.pdf.
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Strunz, Patrick-Pascal Holger. "Interaktion von TRPC-Ionenkanälen mit dem Immunophilin FKBP52." Doctoral thesis, 2020. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-204298.
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Einleitung: TRPC-Kanäle spielen eine wichtige Rolle in der Pathologie der Herzinsuffizienz und kardialen Hypertrophie. Diese Effekte werden unter anderem über den Calcineurin-NFAT-Signalweg vermittelt. Ein wichtiger Interaktionspartner und Regulator von TRPC-Kanälen ist das Protein FKBP52. Mittels eines Yeast Two-Hybrid Systems wurde in einer kardialen cDNA library eine Interaktion zwischen einem C-terminalen Fragment von TRPC3 (AS 742-848), welches außerhalb der bekannten FKBP-Bindungsdomäne (AS 703-714) liegt, und FKBP52 beobachtet. Da dies eine weitere Bindungsstelle in FKBP52 vermuten ließ, erzeugten wir ein Fragment von FKBP52, welches FKBP52s genannt wurde und dem die funktionell relevante PPIase I-Domäne mit der bekannten Bindungsstelle fehlt. Eine erste Co-IP zwischen diesem Fragment und TRPC3 war erfolgreich. Ziel: Die Bestimmung, ob die Anwesenheit des verkürzten FKBP52 in vivo die Komplexbildung aus TRPC3 bzw. TRPC4 und dem Wildtyp-FKBP52 unterdrückt. Zusätzlich, ob FKBP52s die Interaktion zwischen TRPC3 bzw. TRPC4 und Calcineurin in vivo unterbricht und damit die Aktivierung des Calcineurin-NFAT-Signalweges hemmt. Methoden: Co-Immunopräzipitationen (Co-IP) wurden mit HEK-293-Zellen durchgeführt, die mit cDNA transfiziert wurden, welche Gene für TRPC3, TRPC4, Calcineurin A und FKBP52s enthielt. Zur Bestimmung der nukleären Translokation von NFATc1 mittels Fluoreszenzmikroskopie wurden HEK-293-Zellen mit TRPC3, TRPC4, GFP-NFATc1 ± FKBP52s transfiziert. Die statistische Analyse erfolgte mit einer One-Way ANOVA. Ergebnisse: In dieser Arbeit konnte gezeigt werden, dass FKBP52 sowohl mit TRPC3 als auch mit TRPC4 interagiert. Ebenso wurde festgestellt, dass FKBP52 auch ohne seine katalytische PPIase I-Domäne Bindungen mit TRPC3 bzw. TRPC4 eingeht. Dieses FKBP52-Konstrukt nimmt ebenso an der Komplexbildung mit TRPC3 bzw. TRPC4 und Calcineurin teil. Des Weiteren ließ sich für TRPC3 zeigen, dass unter Stimulation mit Carbachol (GPCR-Agonist) bei Anwesenheit dieses gekürzten FKBP52 eine signifikant geringere Aktivierung und Wanderung des Transkriptionsfaktors NFAT in den Nucleus erfolgte. Schlussfolgerung: FKBP52 spielt daher eine wichtige Rolle in dieser Signalkaskade, indem es entscheidend an der Aktivierung von Calcineurin und dessen Rekrutierung zum TRPC-Kanalkomplex beteiligt ist und damit auch an der Aktivierung des Calcineurin-NFAT-SignalwegesBackground: TRPC channels play an important role in the pathology of heart failure and cardiac hypertrophy. These effects are partly mediated by the calcineurin-NFAT signaling pathway. An important binding partner and regulator of TRPC channels is the protein FKBP52. Using a Yeast Two-Hybrid System in a cardiac cDNA library, we have recently found an interaction between a C-terminal fragment of TRPC3 (aa 742-848) without the known FKBP binding site (aa 703-714) and FKBP52 indicating a new binding domain. After creation of a FKBP52 fragment – called FKBP52s -, lacking the functional relevant PPIase domain with the known binding site, a first immunoprecipitation between this fragment and TRPC3 was successful. Aim: To analyze whether the presence of the truncated FKBP52 in vivo suppresses complex formation between TRPC3 or TRPC4 and the wild-type FKBP52. In addition, whether FKBP52s interrupts assembling between TRPC3 or TRPC4 and calcineurin in vivo and so activation of the calcineurin-NFAT pathway. Methods: Co-immunoprecipitation (Co-IP) experiments were performed in HEK 293 cells which were transfected with cDNAs encoding TRPC3, TRPC4, calcineurin A and FKBP52s. For detecting the translocation of NFATc1 into the nucleus by fluorescence microscopy, HEK 293 cells were transfected with TRPC3, TRPC4, GFP-NFATc1 ± FKBP52s. Statistical analysis was performed by one-way ANOVA. Results: In this study it was demonstrated for the first time that FKBP52 interacts not only with TRPC4 but also with TRPC3. Additionally, it was shown that a protein fragment of FKBP52 without its PPIase I domain also binds TRPC3 and TRPC4. This PPIase-deficient FKBP52 protein takes part in complex formation between TPRC3 and TRPC4, respectively, and calcineurin. Furthermore, it was shown for TRPC3 that in presence of this FKBP52 fragment, the activation of calcineurin and nuclear translocation of NFAT was significantly reduced after stimulation with c arbachol (GPCR-agonist). Conclusion: Thus, FKBP52 is important in this signaling cascade by assembling calcineurin to the TRPC channel complex and thus also in the activation of the calcineurin-NFAT signaling pathway
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Germain, Marie-Anne. "Découverte de nouvelles interactions entre le virus de l'Hépatite C et l'hôte par une approche combinée de Spectrométrie de Masse et de Génomique Fonctionnelle." Thèse, 2012. http://hdl.handle.net/1866/10026.
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La réplication et l’assemblage du virus de l’hépatite C (VHC) sont régulés finement dans le temps et l’espace par les interactions protéiques entre le virus avec l’hôte. La compréhension de la biologie du virus ainsi que sa pathogénicité passe par les connaissances relatives aux interactions virus/hôte. Afin d’identifier ces interactions, nous avons exploité une approche d’immunoprécipitation (IP) couplée à une détection par spectrométrie de masse (MS), pour ensuite évaluer le rôle des protéines identifiées dans le cycle viral par une technique de silençage génique.
Les protéines virales Core, NS2, NS3/4A, NS4B, NS5A et NS5B ont été exprimées individuellement dans les cellules humaines 293T et immunoprécipitées afin d’isoler des complexes protéiques qui ont été soumis à l’analyse MS. Ainsi, 98 protéines de l’hôte ont été identifiées avec un enrichissement significatif et illustrant une spécificité d’interaction. L’enrichissement de protéines connues dans la littérature a démontré la force de l’approche, ainsi que la validation de 6 nouvelles interactions virus/hôte. Enfin, le rôle de ces interactants sur la réplication virale a été évalué dans un criblage génomique par ARN interférant (ARNi). Deux systèmes rapporteurs de la réplication virale ont été utilisés : le système de réplicon sous-génomique (Huh7-Con1-Fluc) et le système infectieux (J6/JFH-1/p7Rluc2a), ainsi qu’un essai de toxicité cellulaire (Alamar Blue). Parmi les protéines de l’hôte interagissant avec le VHC, 28 protéines ont démontré un effet significatif sans effet de toxicité cellulaire, suggérant fortement un rôle dans la réplication du VHC.
Globalement, l’étude a mené à l’identification de nouvelles interactions virus/hôte et l’identification de nouvelles cibles thérapeutiques potentielles.Hepatitis C virus (HCV) replication and assembly are tightly regulated in time and space within the cell, most likely due to protein interactions between virus and host. In order to better understand HCV biology and its pathogenesis, there is a need to unravel virus/host interaction network. We extended our knowledge of virus/host interactions by the identification of cellular proteins associated to HCV proteins using an immunoprecipitation (IP) technique coupled to mass spectrometry (MS), and further evaluate the role of retrieved interactors using gene knockdown. FLAG-tagged viral proteins Core, NS2, NS3/4A, NS4B, NS5A and NS5B have been expressed individually in 293T human cells, and immunoprecipitated protein complexes have been submitted to MS analysis for identification of host proteins. In this study, 98 proteins were significantly enriched and showed specific interaction to a viral protein. Retrieval of previously characterized interacting proteins proved the strength of the method. Six newly identified interactors by MS were individually confirmed using IP of viral proteins. We evaluated the role of identified interactors in HCV replication by performing a functional lentivirus-based RNA interference (RNAi) screen. Two reporter systems were used: the sub- genomic replicon (Huh7-Con1-Fluc) and a full length infectious clone (J6/JFH-1/p7Rluc2a), as well as the cellular toxicity assay Alamar blue. Of the identified host interactors, 28 proteins showed a significant effect on HCV replication upon gene knockdown and without cellular toxicity. Overall, the study led to the identification of novel virus/host interactions essential in HCV life cycle and provides novel potential drug targets.
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Hui, Kelvin Kai-Wan. "Analysis of Mitochondrial Signaling in the Regulation of Programmed Cell Death." Thesis, 2011. http://hdl.handle.net/1807/29750.
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The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death.
In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival.
The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.