<p>Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. </p><p>The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.</p><p>In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.</p><p>In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. </p><p>In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.</p>

Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development. In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

No description available.

Aquesta tesi es centra en la caracterització funcional d'una proteïna de xoc de calor de baix pes molecular (Small Heat Shock Protein - sHSP) de classe I de surera pel que fa a la seva capacitat per protegir les cèl·lules de l'estrès i per estabilitzar les membranes. Les sHsps són proteïnes que s'expressen en condicions d'estrès cel·lular. Encara que certs aspectes funcionals de les sHsps són ben coneguts, el nostre treball aporta informacions noves sobre el paper de les diferents regions de la proteïna, especialment de la regió N-terminal.L'objectiu concret d'aquest treball és determinar la funció termoprotectora de QsHsp17.4-CI, una sHsp de classe I oobtinguda a partir de les cèl·lules de fel·lema d'alzina surera, en un model bacterià i analitzar la importància de les diferents regions de la proteïna en aquesta funció. Amb aquesta finalitat s'han dissenyat dues proteïnes parcials derivades de QsHsp17.4-CI: una a la que li falta la regió N-terminal (C105) i una altra amb pràcticament tot el domini &#61537;-cristal·lí deleccionat (N61), i una tercera, derivada de QsHs10-CI, a la que li falta la meitat del domini &#61537;-cristal·lí (Hsp10). També s'estudia la possible capacitat estabilitzadora de membranes i la capacitat de modificar l'expressió d'altres Hsps quan s'expressa de forma heteròloga.Els nostres resultats demostren que l'expressió de QsHsp17.4-CI protegeix a les cèl·lules d'E.coli de l'estrès tèrmic alhora que la regió N-terminal i la regió consens II del domini &#61537;-cristal·lí són imprescindibles per aquesta funció de protecció. En relació a un possible paper en les membranes, els estudis de localització subcel·lular mostren que QsHsp17.4-CI colocalitza amb la fracció membranes i que la regió N-terminal de la proteïna és responsable d'aquesta colocalització. No s'ha pogut demostrar, però, que la localització amb la membrana estigui associada a un efecte protector d'aquesta: en cap cas la sobrexpressió de les proteïnes modifica la composició d'àcids grassos i només N61, que no té acció termoprotectora, altera l'estat fisico-químic de la membrana. En estudis d'expressió de novo en E.coli s'ha observat que, a diferència de les altres proteïnes heteròlogues, N61 activa l'expressió de la majoria de Hsps d'E.coli fent pensar en una possible relació entre l'estat físic de la membrana i l'activació de la resposta a l'estrès.En resum, en aquest treball hem provat la capacitat protectora de QsHsp17.4 i aportem noves dades sobre la importància de la regió N-terminal i la regió consens II del domini &#61537;-cristal·lí en aquesta funció. Per altra banda, es suggereix que QsHsp17.4 podria interaccionar amb la membrana d'E.coli i que la regió N-terminal seria imprescindible per aquesta interacció. Finalment hem determinat que les proteïnes que provoquen variacions en l'estat de fluïdesa de la membrana poden activar la resposta al xoc de calor per part de la cèl·lula bacteriana. / This thesis is focused in the functional studies of a Small Heat Shock Protein (sHsp). sHsps are expressed under stress conditions. Although some functional aspects of these proteins are known, our work aport new data about the role of the different protein regions, especially the N-terminal region. The aim of this work is to demonstrate a thermotolerance effect of QsHsp17.4-CI in bacterial cells and to analyze the importance of the protein regions in this function. To achieve this objective two deletion mutants derived from QsHsp17.4-CI were designed: a protein lacking the N-terminal region (C105) and a protein where the entire &#61537;-cristallin domain is missing (N61) and a third mutant, derived from QsHsp10-CI, that bears half of the &#61537;-cristallin domain (Hsp10). To better understand the functional mechanism of sHsps we study the membrane stabilizing capacity of QsHsp17.4-CI as well as its capacity to modify other Hsps expression.Our results demonstrate that the expression of QsHsp17.4-CI protects E.coli cells from a heat shock and that the N-terminal region and the consensus region II of the &#61537;-cristallin domain are necessary for this protective function. Related to a possible role in membranes, location studies suggest that QsHsp17.4-CI colocalizes with cell membrane fraction and that N-terminal region is important for this location. However, no relation between membrane localization and a protective effect has been demonstrated: Protein overexpression does not modify membrane fatty acid composition and only N61, which has no thermoprotection, changes membrane physical state. Studies of E.coli de novo synthesis show that, unlike the other recombinant proteins, the overexpression of N61 activates the expression of almost all E.coli Hsps suggesting a possible relation between membrane physical state and the activation of the heat shock response.As summary, in this work we have demonstrated the thermoprotective capacity of QsHsp17.4-CI and we contribute with new data about the importance of N-terminal region and consensus region II of &#61537;-cristallin domain for this function. On the other hand, we suggest the possibility that QsHsp17.4-CI interacts with membrane and that N-terminal region is important for this interaction. Lastly, we have observed how changes in membranes fluidity state can activate heat shock response in bacterial cells.

No description available.

Les glycosidases sont des enzymes impliquées dans de nombreux processus biologiques. Entre autres, elles sont responsables de la dégradation des déchets polysaccharidiques de nos cellules. Lorsqu’une modification génétique touche un gène qui code pour une de ces enzymes, des pathologies graves regroupées sous l’appellation de « maladies lysosomales » peuvent être déclenchées. L'objectif de ce projet a été de proposer une méthode de synthèse efficace de molécules potentiellement actives spécifiquement sur l'une ou l'autre de ces maladies. Les molécules ciblées sont des inhibiteurs de glycosidases de la famille des aminocyclitols, utilisés dans une stratégie thérapeutique émergente « par molécules chaperonnes ». La méthode de synthèse développée s’appuie sur une étape enzymatique clé utilisant les aldolases comme catalyseurs et répondant aux contraintes environnementales actuelles de la chimie verte. Nous avons atteint nos objectifs grâce à l’utilisation de trois aldolases différentes, produites et purifiées pour la première fois au sein de notre laboratoire. Il s’agit de la fuculose-1-phosphate aldolase F1PA, de la rhamnulose-1-phosphate aldolase R1PA et de la nouvellement découverte fructose-6-phosphate aldolase FSA. La formation d’une quarantaine de nitrocyclitols, de stéréochimies définies, précurseurs des aminocyclitols correspondant, a ainsi été réalisée avec de très bons rendements de synthèse. / Glycosidases are enzymes involved in many biological processes. For example, they are responsible for breaking up polysaccharide waste materials of our cells. When a genetic mutation concerns a gene encoding for one of theses enzymes, acute pathologies named lysosomal storage disorders can appear. Aim of this work was to find an effective synthesis method of molecules potentially active specifically on one or others diseases. Target molecules are glycosidases inhibitors from the aminocyclitols family, used in an emergent strategy “by molecular chaperones”. The method of synthesis developed in the course of this work is based on an enzymatic key step using aldolases as catalyst, and follows current environment constraints of the green chemistry concept. Goals were reached thanks to the use of three different aldolases, produced and purified for the first time in our lab. It consists in fuculose-1-phosphate aldolase F1PA, rhamnulose-1-phosphate aldolase R1PA and the newly discovered fructose-6-phosphate aldolase FSA. Formation of around forty nitrocyclitols (aminocyclitols precursors) with a defined stereochemistry was realised with very good yields of synthesis.

Abstract The opioid receptors (&#948;, &#954; and &#956;) are family A G protein-coupled receptors (GPCRs) that have an important role in the regulation of pain. Like all GPCRs they have a common structure that consists of seven transmembrane domains with an extracellular amino (N)-terminus and an intracellular carboxyl-terminus. The human &#948; opioid receptor (h(&#948;OR) has two polymorphic variants. A single-nucleotide polymorphism causes replacement of Phe with Cys at the amino acid position 27 in the receptor N-terminus. The allelic frequency of h&#948;ORCys27, the less common variant, is about 10% in Caucasians. In this study, the two h&#948;OR variants were expressed in heterologous expression systems and their biosynthesis was characterized in detail using various cell biological and biochemical techniques. In particular, the role of receptor heteromerization and opioid receptor pharmacological chaperones in processing, maturation and trafficking of the variants was assessed. The h&#948;OR variants showed significant differences in maturation and trafficking. The h&#948;ORCys27 had a significantly lower maturation efficiency compared with h&#948;ORPhe27. In addition, long-term receptor expression led to the accumulation of h&#948;ORCys27 in the endoplasmic reticulum (ER) and also impaired receptor targeting to ER-associated degradation. The h&#948;OR variants also differed at the cell surface, as the h&#948;ORCys27 variant was internalized constitutively in a faster and more extensive manner than h&#948;ORPhe27. However, the variants had similar pharmacological properties and activated G proteins in an identical manner. This study also showed that h&#948;ORCys27 acted in a dominant negative manner and redirected some h&#948;ORPhe27 precursors to degradation. This resulted in impaired plasma membrane expression of h&#948;ORPhe27 in co-transfected cells. The h&#948;OR variants were found to form heteromers early in the secretory pathway, which is the most likely reason for the dominant negative behavior of h&#948;ORCys27 on h&#948;ORPhe27. The mechanism of action of opioid receptor pharmacological chaperones, membrane-permeable opioid ligands, was investigated in detail using h&#948;ORCys27 and its mutant form h&#948;ORCys27-(Asp95Ala) as models. Opioid antagonists were found to be able to bind to and stabilize receptor precursors in the ER and enhance their dissociation from the ER molecular chaperone calnexin. This led to an increase in the number of receptors at the plasma membrane. In addition, h&#948;ORPhe27, like h&#948;ORCys27, was responsive to antagonist treatment whether the variants were expressed together or individually. / Tiivistelmä Opioidireseptorit kuuluvat G-proteiinikytkentäisiin reseptoreihin, ja niillä on tärkeä rooli kipuaistimuksen säätelyssä. Ne ovat solukalvoproteiineja, joiden aminohappoketju läpäisee kalvon seitsemän kertaa. Reseptorien aminoterminaalipää sijaitsee solun ulkopuolella ja karboksiterminaalipää solun sisällä. Ihmisen &#948;-opioidireseptori esiintyy kahtena polymorfisena muotona, Phe27:nä ja Cys27:nä, joissa aminohappo 27 on joko fenyylialaniini (Phe) tai kysteiini (Cys). Cys27 on harvinaisempi muoto, ja sen yleisyys on noin 10 % eurooppalaista alkuperää olevalla väestöllä. Tämän väitöskirjan tavoitteena oli tutkia &#948;-opioidireseptorin varianttimuotojen biosynteesiä reseptoriproteiinia tuottavissa heterologisissa solumalleissa (HEK293- ja SH-SY5Y-solut) solubiologisilla ja biokemiallisilla menetelmillä.. Väitöskirja osoittaa, että &#948;-opioidireseptorin varianttimuotojen välillä on eroa prosessoinnissa. Cys27-varianttia kuljetetaan endoplasmakalvostosta solun pinnalle vähemmän kuin Phe27-varianttia, ja pitkäaikainen reseptorituotanto johtaa vastasyntetisoituneiden reseptorien kerääntymiseen solun sisälle. Samalla reseptorien ohjaus proteasomihajotukseen heikkenee. Soluissa, jotka tuottavat molempia varianttimuotoja samanaikaisesti, Cys27-variantin havaittiin ohjaavan myös Phe27-varianttia proteasomihajotukseen vähentäen sen kuljetusta solun pinnalle. Tämä Cys27-variantin dominanttinegatiivinen ominaisuus johtuu todennäköisesti siitä, että variantit muodostavat dimeerisen rakenteen endoplasmakalvostossa. Havaittiin myös, että Cys27-varianttireseptorit ohjataan solun pinnalta lysosomihajotukseen tehokkaammin kuin vastaavat Phe27-varianttimuodot. Prosessointieroista huolimatta variantit eivät poikkea toisistaan farmakologisilta ominaisuuksiltaan, ja ne aktivoivat G proteiineja samalla tavalla. Väitöskirjassa tutkittiin myös farmakologisten kaperonien toimintamekanismeja käyttämällä mallina &#948;-opioidireseptorin Cys27-varianttia ja sen pistemutaatiota (Asp95Ala). Farmakologisten kaperonien eli reseptorispesifisten ligandien todettiin sitoutuvan reseptoreihin endoplasmakalvostossa ja stabiloivan niiden rakennetta, mikä vähentää reseptorin ja proteiinien laadunvalvontaan osallistuvan kaperonin, kalneksiinin, välistä vuorovaikutusta. Tämä johtaa reseptorien määrän kasvuun solun pinnalla.

Molecular chaperones are a group of proteins which maintain cellular homeostasis by assisting de novo protein folding and their refolding to native state after destabilization due to external stress. They are also known as heat shock proteins as they were first discovered as a response to heat stress. It is now well established that the function of this group of proteins is not only restricted to protein homeostasis but also extends to diverse cellular processes such signal transduction, development and differentiation. Heat shock protein 90 (Hsp90) is one of the most abundant molecular chaperones that is highly conserved from prokaryotes to eukaryotes. Hsp90 is an essential chaperone and is required for the viability of all eukaryotes examined so far including yeast, Drosophila and Caenorhabditis elegans. Hsp90 has emerged as an important regulator of cellular activities by virtue of its ability to interact with a diverse set of client proteins many of which include transcription factors, protein kinases and signaling molecules. Through interaction with these proteins it is involved in regulating cellular processes including growth, cell cycle, endocrine functions, apoptosis, differentiation and development. Further in Drosophila and plants, Hsp90 is thought to function as a capacitor for morphological evolution and phenotypic variation. Recently, it has also been implicated in the emergence of drug resistance in Candida albicans. Furthermore, the importance of Hsp90 in disease states, particularly in cancer, is strongly evident, where chaperoning of mutated and oncogenic proteins is critical for continuous proliferation of cells. This has led to the development of Hsp90 inhibitors as an anti-cancer drug. Geldanamycin (GA), a benzoquinone ansamycin was the first molecule shown to inhibit Hsp90 activity by binding to its ATP binding domain. A derivative of GA, 17-allylamino-17-demethoxygeldanamycin (17AAG), has shown promise in clinical studies and has entered Phase III clinical trials. Hsp90 has been shown to be important for growth and development of many protozoan parasites. Inhibition of Hsp90 function in Leishmania, Emiera, Toxoplasma, Trypanosoma as well as Plasmodium causes a block in their developmental cycle. Previous studies from our laboratory have shown that inhibition of Hsp90 function prevents growth of malaria parasite in human erythrocytes in vitro. P. falciparum Hsp90 (PfHsp90) has also been shown to regulate parasite growth during the febrile episodes that are characteristic of malaria. While most of the studies highlighting the importance of PfHsp90 have relied on its pharmacological inhibition, its biochemical characterization and quantitative measurement of its interaction with GA in isolated system has not been explored. It was also not understood whether the in vitro model of Hsp90 inhibition could translate into inhibition of the parasite growth in an animal model of malaria. Since Hsp90 is a split ATPase requiring proper co-ordination between the residues on its N-terminal and middle domains, it would be desirable to biochemically characterize full length PfHsp90 to gain insights into its potential as an anti-malarial target. The present study was initiated with an objective of understanding the biochemical properties of Hsp90 from P. falciparum in terms of ATP binding, ATP hydrolysis and its GA binding ability. We have also examined the potential of PfHsp90 to serve as a chemotherapeutic target using its clinically well-established inhibitor, 17AAG, in a preclinical mice model. Apart from using in vitro and in vivo models of malaria, we have also explored the efficacy of 17AAG in the P. falciparum samples collected from malaria patients. Additionally, we have examined the relevance of chaperones, in particular PfHsp90 in the samples collected from malaria patients. Finally, we have attempted to understand the unexplored biology of another malaria parasite P. vivax by a high throughput proteomics approach. Biochemical characterization of PfHsp90 and its comparison with host Hsp90 Hsp90 belongs to GHKL (gyrase, Hsp90, histidine kinase, MutL) protein family having a characteristic novel ATP-binding Bergerat fold. The ATP binding pocket of GHKL family differs from the conventional nucleotide binding fold in the formation of a cone shaped pocket made up of four anti-parallel β-sheets and three α helices as opposed to parallel βsheets surrounded by α-helices in the latter. The most distinctive feature of Bergerat fold is the presence of ATP lid. Further, even within the GHKL family members the composition and the conformation of this ATP-lid differs, leading to different solvent exposure of the bound ATP. All Hsp90s from different organisms, characterized so far, have been shown to posses ATP binding and hydrolysis activity but so far PfHsp90 ATPase activity has not been characterized. Using intrinsic tryptophan fluorescence measurements, we found PfHsp90 to bind ATP with about 30% higher affinity than human Hsp90 (hHsp90). We further, 32 determined the ATPase activity of PfHsp90 by monitoring the direct conversion of (γ-P) 32-2 ATP to Pi. PfHsp90 bound and hydrolyzed ATP with a Km of 611 µM and kcat of 9.9 x 10 -1m . Interestingly, PfHsp90 showed six times higher ATPase activity as compared to its human homologue and more intriguingly the ATPase activity exhibited by PfHsp90 was highest among all the Hsp90s studied so far. Previous studies from our laboratory have provided sufficient evidence for inhibitory action of GA on Plasmodium growth inside the infected erythrocytes. GA is known to exert its inhibitory effect by binding to the ATP binding domain of Hsp90 thus inhibiting its chaperone activity. Earlier reports have shown that despite a high similarity between the ATP/GA binding region in Hsp90 from different organisms, there is a difference in their ability to bind GA. For example, in spite of all the hallmarks of ATP-binding pocket of Hsp90 family C. elegans Hsp90 does not bind GA. We have employed fluorescence spectroscopy to examine whether PfHsp90 can bind to GA. In parallel, we have also determined the binding affinity of human Hsp90 (hHsp90) to GA. We observed small but reproducible differences in the binding affinity of GA to Hsp90s from human host and P. falciparum with latter having fourfold higher affinity. A sequence analysis of the GA binding domain of Hsp90s from P. falciparum and human host showed a homologous substitution of K112 of hHsp90 to R98 in PfHsp90. In order to examine the effect of this substitution, if any, on the observed difference in GA binding abilities, we mutated R98 to K in PfHsp90. However, we did not find any difference in the binding ability of R98K PfHsp90 to GA, suggesting that this homologous substitution has minimal or no effect on drug protein interaction in vitro. However, in view of this phylogenetically conserved substitution, we cannot rule out its role in vivo. The chaperone function of Hsp90 is dependent on its ATPase activity which is susceptible to GA mediated inhibition. We next examined the extent of inhibition of GA on the ATPase activity of Hsp90s from P. falciparum and human host. Interestingly, we found the PfHsp90-ATPase activity to be three times more sensitive than hHsp90-ATPase activity to GA mediated inhibition suggesting that the malaria parasite, P. falciparum is likely to be more sensitive to GA when compared to human host. This result is in accordance with a recent study, which has shown that yeast expressing PfHsp90 in lieu of native yeast Hsp90 was more sensitive to GA than yeast expressing either yeast Hsp90 or human Hsp90. Acetylation of Plasmodium falciparum Hsp90 Post-translational modification of Hsp90 such as acetylation has been shown to affect its binding with GA. We first examined whether, PfHsp90 can be acetylated. With the use of various purified Histone acetyl transferases (HATs) of human origin, we have shown PfHsp90 to undergo acetylation in vitro. We found that among different HATs (pCAF, Gcn5 and p300) used, only p300 was able to acetylate PfHsp90 suggesting a role for it in PfHsp90 in vivo acetylation as well. We next examined the in vivo acetylation status of PfHsp90 from parasite lysate. To enrich the acetylated fraction of PfHsp90, we have used Histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Immunoprecipitation of PfHsp90 followed by immunoblotting with an acetyl-lysine antibody confirmed that PfHsp90 undergoes acetylation in vivo. In order to identify the lysine residues which underwent acetylation we subjected the acetylation enriched fraction of PfHsp90 to in-gel trypsin digestion followed by mass spectrometry. Analysis of trypsin digested PfHsp90 from the parasites identified three sites of acetylation, one of which overlapped with PfHsp90 cochaperone (Aha1 and p23) binding residue, suggesting that acetylation could play a potential role in modulating PfHsp90 multi-chaperone complex assembly. Indeed, treatment of P. falciparum cultures with a HDAC-inhibitor resulted in partial dissociation of PfHsp90 complex as observed from size-exclusion chromatography. Adding to this observation, we also found that co-treatment of TSA and GA showed a synergistic and additive effect in inhibiting parasite growth in vitro. The above results suggest the possibility of using Hsp90 inhibitor in combination with HDAC inhibitor to arrest Plasmodium growth and development. Clinically tested GA-analogue 17AAG inhibits Plasmodium growth in vitro and in vivo The specificity of GA inside the cell has been a matter of debate since the discovery of its medicinal importance. In the past, Hsp90 has been implicated as a target of GA by carrying out immunoblotting of GA pull-down fraction with an anti-Hsp90 antibody. Crystal structure of GA with yeast Hsp90 has shown it to bind within the well conserved ATP-binding pocket of Hsp90. However, the specificity of GA inside the cell is still a conjecture. We have performed GA pull down assays from the parasite lysate followed by Coomassie Blue staining, which gave a single band corresponding to 86 kDa PfHsp90. The identity of PfHsp90 was further confirmed by immunoblotting with antibody specific to PfHsp90. This result indicates that inside the cells, inhibitory effect of GA is mediated by and large through its interaction with Hsp90. However, we cannot rule out the presence of other minor, less significant, interactors of GA. Earlier work from our laboratory has shown that GA inhibits Plasmodium growth inside the infected erythrocytes. However, issues related to GA toxicity have excluded its development as a therapeutic. Nevertheless, interest in this class of molecule has led to the generation of a large number of less toxic derivatives of GA. One classical example is 17AAG which has gained clinical importance over the years and has entered in phase III trial. Intrigued by the clinical success of 17AAG, we were interested in determining its ability to modulate parasite growth. Indeed, 17AAG was able to inhibit parasite growth in a manner similar to that of GA. We further extended our study to parasites isolated from patient samples. Here too, we found 17AAG to be effective in inhibiting growth of the parasite. Finally, we examined the efficacy of 17AAG at a pre-clinical level using a mouse model of malaria. Using Peters’ four-day test we found 17AAG, to be effective in attenuating parasite growth and prolonging the survival of parasite infected mice (n=4, p=0.00692; n=10, p=0.001). Clinical relevance of heat shock proteins of Plasmodium falciparum A recent study using in vivo expression profiles of parasites derived from blood samples of infected patients has revealed previously unknown physiological diversity in the biology of malaria parasites. According to gene expression profiles, parasites were clustered into three different physiological states – starvation, glycolysis dependent active growth and environmental stress response. In order to examine the clinical relevance of molecular chaperones in malaria, we reanalyzed the previously published gene expression data of clinical parasites from 46 patients. Our analysis of this data showed that organellar chaperones were up-regulated upon starvation (cluster1) while cytosolic chaperones such as Hsp90 were up-regulated in active growth conditions (cluster2) indicating up-regulation of distinct group of Hsps in response to different environmental cues. Interestingly, Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, clustered in the same group. Further, some patients of cluster 3 (environmental stress response) showed higher expression of Hsp90 while others showed lower expression. In general, cluster 3 group of patients were heterogeneous in terms of expression of chaperones. Using non-negative matrix factorization (NMF), cluster 3 was sub-clustered into two groups 3a and 3b. Cluster 3b showed up-regulation of cytosolic chaperones similar to cluster 2 indicating these two clusters are inter-related. Most of the Hsp90 dependent pathways such as trafficking, signaling, anti apoptotic and pro-survival found to be most active in cluster 2 indicating the dependence of this group of parasites on Hsp90. The two main outcomes of our chaperone analysis are (1) the up-regulation of molecular chaperones in parasites are not a general response to hostile conditions as perceived previously, but is largely determined by the host factors and may differ from one host to another (2) the disease specific pathways may exist in natural condition by the up-regulation of specific chaperone and its interactors as a response to different host environment. Clinical proteomics of human malarial parasites Much of our understanding about the life cycle of parasites and importance of parasite proteins have been gleaned from the studies in laboratory strain or with the laboratory adapted clinical parasites. Although, these studies provide us first hand information about the functionality and the importance of these proteins, but they often fail to mimic the actual disease environment. In the patient, parasites are exposed to host factors such as hormones, metabolites, inflammatory mediators which can influence the expression of proteins and thus parasite biology. Further, instead of parasite exposure to 37°C temperature throughout the erythrocytic cycle in vitro, it is exposed to several rounds of febrile episodes inside human, which can also influence the parasite life cycle. Furthermore, clinical analysis is important to validate the presence and expression of drug targets in actual disease environment. Therefore, analysis of malaria parasite from clinical settings has become an important component in our laboratory and this thesis. Proteomic analysis of clinical samples has emerged as an important tool to understand the proteins dynamicity as response to disease environment. We have initiated clinical proteomic study of P. falciparum, the cause of most common and fatal malaria in humans and extended it further to the neglected malaria parasite P. vivax. The study of P. vivax has largely been over-shadowed by the enormous attention devoted to P. falciparum. Notably, the drugs which have been discovered against P. falciparum are not as effective against P. vivax. Further several unique features of P. vivax such as dormant hyponozoites, reticulocyte host preference and formation of specialized caveolae vesicle complex structure distinguish its biology from P. falciparum and warrant concerted effort directed at this parasite. A major limitation in studying this parasite is the absence of a long-term culturing system. Therefore, research on this parasite requires samples obtained directly from patients. In spite of the inherent difficulty in obtaining such samples, this method provides us an opportunity to study this parasite in its real environment which has a huge effect on the expression as well as function of parasites and host proteins. Our current knowledge about the life cycle of this parasite has been gained from the recently published transcriptome study. Even though transcriptome analyses provide useful understanding at the level of gene expression, they do not reflect the active protein component of a cell. In other words, most of disease outcome is a result of interaction of the protein component with the environment. We therefore attempted to understand the protein component of this parasite in the disease environment to shed light on its pathogenicity. Despite facing several challenges in the way of proteomic analysis of this parasite such as availability of samples, low parasitemia, contamination of parasite proteins with abundant host proteins etc, we were able to identify 154 P. vivax proteins abundantly expressed in clinical environment using mass-spectrometry based approach. We found many proteins unique to this parasite along with known drug targets. This study is the first of its kind and could prove to be a very important step towards gaining insights into the physiology of this parasite.This study serves as a proof-of-principle method which in future is likely to help in identifying many more potential drug targets, vaccine candidates and diagnostic markers from clinically relevant samples as opposed to cultured samples. Summary Despite the importance of PfHsp90 in malaria biology, it has not been characterized in terms of its biochemical properties and its interaction with the inhibitor. In this study, we have successfully cloned, expressed, purified and characterized full length PfHsp90. We found that PfHsp90 exhibits a hyper-ATPase activity and is more sensitive to GA mediated inhibition as compared to human Hsp90. We have also shown that its sensitivity towards GA is dependent on its acetylation status as treatment of infected erythrocytes with HDAC inhibitors increases its sensitivity to GA. Using a pull-down assay, we have determined, unequivocally, that GA specifically binds to Hsp90. Most importantly, we have demonstrated that 17AAG, a clinically well-established inhibitor of Hsp90, inhibits parasite growth in a laboratory strain, field isolates and an in vivo mouse model of malaria. Overall, our biochemical characterization and drug interaction studies underscore the importance of PfHsp90 as a potent drug target and its inhibitors as a candidate drugs for the treatment of malaria, one of the deadly human infectious diseases. Our efforts to understand the importance of molecular chaperones in parasites isolated directly from patient samples (clinical setting) has revealed conspicuous association of Hsps with previously defined parasite physiological states. In particular, parasites obtained from a specific group of patients exhibited heightened dependence on Hsp90-dependent pro-survival pathways, indicating an increased response to host stressors in this group of parasites. Thus, parasite encoded chaperones, in particular PfHsp90, play a major role in defining the pathogenesis of malaria. A disease is an outcome of interaction between pathogens and its host, therefore it is important to study parasite in its real environment to understand disease pathogenesis. Our lab has previously reported the first ever proteomic analysis of P. falciparum from malaria patients. In this study, we have made an attempt to understand the unexplored biology of another important malaria parasite P. vivax. We have used a mass-spectrometry based approach to identify the protein content of this parasite. This technically challenging attempt has enabled us to identify many proteins. This study is an important step towards understanding the biology of this parasite in dearth of any information available on the proteins involved in this parasite’s pathogenicity.