From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

No description available.

Acyl-CoA binding protein (ACBP) ubiquitously found in eukaryotic organisms fulfills important functions of solubilisation, protection and transport of acyl-CoA esters, a major intermediate of lipid metabolism. This thesis presents an investigation of the physiological role of the small cytosolic ACBP in seed oil biosynthesis. The second important objective of this study was to evaluate the use of ACBP as a molecular tool for modification of seed oil content and/or fatty acid (FA) composition. Agrobacterium-mediated transformation of Arabidopsis thaliana and Brassica napus was performed with a number of genetic constructs designed for seed-specific expression of the B. napus cDNA encoding a small cytosolic ACBP. Protein level and subcellular localization of BnACBP in A. thaliana transgenic seeds depended on the structure of the genetic constructs mainly, the presence of additional in-frame sequences, encoding a protein fusion partners or signal peptides. Seed oil from A. thaliana T2 and T3 seeds had increased polyunsaturated fatty acid (PUFA) percentage (18:2cis delta9,12 and, in some lines, 18:3cis delta9,12,15) at the expense of very-long-chain monounsaturated (20:1cis delta11) and saturated (18:0) fatty acids. An increase in PUFA levels in seed oil was due to enhanced acyl channeling from the acyl-CoA pool to phosphatidylcholine, the substrate for extraplastidial FA desaturation. The activity of A. thaliana acyl-CoA: lysophosphatidylcholine acyltransferase (AthLPCAT), an enzyme involved in acyl exchange between acyl-CoA and PC, was significantly increased in the presence of the recombinant B. napus ACBP (rBnACBP) in the reaction mixture. rBnACBP also modulated enzymatic activities of glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase in vitro. Finally, the effect of constitutive or seed-specific gene silencing of ACBP on seed oil formation was examined. A. thaliana transformation with RNAi constructs resulted in partial suppression of ACBP expression and changes in FA composition of seed oil which included an increase in the percentage of 18:1cis delta9 and 18:2cis delta9,12 and, decrease of 18:3cis delta9,12,15. Overall, the results of this study demonstrate that the small cytosolic ACBP plays an important role in acyl exchange between acyl-CoA and PC metabolic pools. Overexpression of ACBP during seed development can be useful in genetic engineering strategies aimed at modifying the FA composition of seed oils. / Plant Science

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

High-value industrial oils are essential for many processes and have great economic and environmental impacts. The tung tree produces a high-value seed oil. Approximately 80% of tung oil is α-eleostearic acid, which has a high degree of unsaturation thus giving it properties as a drying oil. The identification of the biological components in tung is imperative to further the knowledge of its processes. Four unique tung acyl-CoA binding proteins, VfACBP3a, VfACBP3b, VfACBP4, and VfACBP6 were identified and the genes encoding them were cloned and analyzed to determine their biological roles. The VfACBPs were observed to be similar to other organisms' ACBPs, especially Arabidopsis thaliana. In addition, each gene was expressed in all tung tissues. They were shown to interact with VfDGAT1 and VfDGAT2, two known components of tung lipid metabolism. Finally, VfACBP3a and VfACBP6 were expressed in the seeds of transgenic plants to study the effects of VfACBP expression on seed lipid fatty acid content.

Neste trabalho foi utilizado um esquema multi-técnicas para estudar a base de interações moleculares protagonizadas por duas proteínas que possuem funções biológicas completamente distintas. A primeira delas, clorocatecol 1,2-dioxigenase (Pp 1,2-CCD), tem um apelo biotecnológico para área ambiental devido a sua capacidade de catalisar a degradação do composto clorocatecol, um intermediário comum no final da decomposição de diversos hidrocarbonetos aromáticos policíclicos. Essa característica pode promover a descontaminação de solos e águas poluídos revelando um grande potencial para aplicações em mecanismos de biorremediação. Além disso, a presença de moléculas anfipáticas junto à interface de ligação dos monômeros da CCD levantou a questão em relação à capacidade dessa família de enzimas de se ligar a membranas biológicas. Esse tipo de informação amplia o conhecimento acerca de mecanismos básicos de ação da enzima, aumentando a possibilidade de parceiros de interação, podendo levar a outras formas de controle da atividade biológica para uso em aplicações biotecnológicas como desenvolvimento de biossensores. O estudo dessa enzima está, portanto, voltado para a compreensão de suas interações com miméticos de membrana e a tentativas de imobilização da proteína nestas estruturas. Para isto, fazemos uso de técnicas biofísicas como dicroísmo circular, caloria diferencial de varredura e espectroscopias ópticas, e biomoleculares como desenvolvimento de oligonucleotídeos, reações de cadeia polimerase e análise de restrição. A outra vertente desta dissertação, tem como foco de estudo a proteína ligante de acil-CoA de Cryptococcus neoformans (CnACBP) clonada pela primeira vez em nosso laboratório. Homólogos de ACBP foram encontrados em todos os organismos distribuídos nos quatro reinos eucariotos, com alta similaridade sequencial (~48%). A sua presença ao longo dos reinos e seu envolvimento em diversos mecanismos metabólicos essenciais relacionados ao éster acil-CoA levaram à conclusão de que se trata de uma housekeeping protein, e não uma proteína específica, confinada a um tipo especializado de célula. Este trabalho traz uma caracterização inicial da CnACBP que busca esclarecer questões ainda em aberto e também aprofundar o conhecimento ainda muito vago de como cargas e a presença do ligante podem influenciar estrutura, estabilidade e função através de técnicas termodinâmicas e espectroscópicas antes de um aprofundamento de seu papel no interior da célula e interações com outras proteínas. / In this study, we used a multi technique approach to understand the basic molecular interactions of two proteins that have quite different biological functions. The first, chlorocatechol 1,2- dioxygenase (Pp 1,2-CCD), has an environmental appeal due to it ability to catalyze the degradation of chlorocatechol, a common intermediate in the end of the decomposition of many polycyclic aromatic hydrocarbons. This characteristic of decontaminating polluted soils and waters suggest a great potential for applications in bioremediation mechanisms. Moreover, the presence of amphipathic molecules at the interface of the CCD monomers raised issues related to the ability of this enzyme family of binding to biological membranes. Such information broadens the knowledge of the basic mechanisms of enzyme action, increasing the possibility of interaction partners and may lead to other forms of control of the biological activity for use in biotechnological applications, such as biosensors development. The study of this enzyme is therefore, aimed at understanding their interactions with mimetic membrane and immobilization attempts of the protein in these structures. For this purpose, we make use of biophysical techniques such as circular dichroism, differential scanning calorimetry and optical spectroscopies and biomolecular techniques, such as development of primers, polymerase chain reaction and restriction analysis. The other aspect of this dissertation is focused on the study of acyl-CoA binding protein of Cryptococcus neoformans (CnACBP) cloned for first time in our laboratory. Homologues of ACBP were found in all organisms distributed in the four kingdoms of eukaryotes, with high sequence similarity (~ 48%). Its widespread presence and their involvement in several key metabolic pathways related to the acyl-CoA ester led to the conclusion that ACBP is a housekeeping protein and not a specific protein contained a specialized cell type. Here we present an initial characterization of CnACBP that seeks to relevant issues regarding the proteins function. Our goal was to increase the still vague knowledge on how electrical charges and the presence of the binding partner may influence the structure, stability and function through thermodynamic and spectroscopic techniques. This is an initial step toward the full understanding of the role of protein in the cell.

Neste trabalho foi utilizado um esquema multi-técnicas para estudar a base de interações moleculares protagonizadas por duas proteínas que possuem funções biológicas completamente distintas. A primeira delas, clorocatecol 1,2-dioxigenase (Pp 1,2-CCD), tem um apelo biotecnológico para área ambiental devido a sua capacidade de catalisar a degradação do composto clorocatecol, um intermediário comum no final da decomposição de diversos hidrocarbonetos aromáticos policíclicos. Essa característica pode promover a descontaminação de solos e águas poluídos revelando um grande potencial para aplicações em mecanismos de biorremediação. Além disso, a presença de moléculas anfipáticas junto à interface de ligação dos monômeros da CCD levantou a questão em relação à capacidade dessa família de enzimas de se ligar a membranas biológicas. Esse tipo de informação amplia o conhecimento acerca de mecanismos básicos de ação da enzima, aumentando a possibilidade de parceiros de interação, podendo levar a outras formas de controle da atividade biológica para uso em aplicações biotecnológicas como desenvolvimento de biossensores. O estudo dessa enzima está, portanto, voltado para a compreensão de suas interações com miméticos de membrana e a tentativas de imobilização da proteína nestas estruturas. Para isto, fazemos uso de técnicas biofísicas como dicroísmo circular, caloria diferencial de varredura e espectroscopias ópticas, e biomoleculares como desenvolvimento de oligonucleotídeos, reações de cadeia polimerase e análise de restrição. A outra vertente desta dissertação, tem como foco de estudo a proteína ligante de acil-CoA de Cryptococcus neoformans (CnACBP) clonada pela primeira vez em nosso laboratório. Homólogos de ACBP foram encontrados em todos os organismos distribuídos nos quatro reinos eucariotos, com alta similaridade sequencial (~48%). A sua presença ao longo dos reinos e seu envolvimento em diversos mecanismos metabólicos essenciais relacionados ao éster acil-CoA levaram à conclusão de que se trata de uma housekeeping protein, e não uma proteína específica, confinada a um tipo especializado de célula. Este trabalho traz uma caracterização inicial da CnACBP que busca esclarecer questões ainda em aberto e também aprofundar o conhecimento ainda muito vago de como cargas e a presença do ligante podem influenciar estrutura, estabilidade e função através de técnicas termodinâmicas e espectroscópicas antes de um aprofundamento de seu papel no interior da célula e interações com outras proteínas. / In this study, we used a multi technique approach to understand the basic molecular interactions of two proteins that have quite different biological functions. The first, chlorocatechol 1,2- dioxygenase (Pp 1,2-CCD), has an environmental appeal due to it ability to catalyze the degradation of chlorocatechol, a common intermediate in the end of the decomposition of many polycyclic aromatic hydrocarbons. This characteristic of decontaminating polluted soils and waters suggest a great potential for applications in bioremediation mechanisms. Moreover, the presence of amphipathic molecules at the interface of the CCD monomers raised issues related to the ability of this enzyme family of binding to biological membranes. Such information broadens the knowledge of the basic mechanisms of enzyme action, increasing the possibility of interaction partners and may lead to other forms of control of the biological activity for use in biotechnological applications, such as biosensors development. The study of this enzyme is therefore, aimed at understanding their interactions with mimetic membrane and immobilization attempts of the protein in these structures. For this purpose, we make use of biophysical techniques such as circular dichroism, differential scanning calorimetry and optical spectroscopies and biomolecular techniques, such as development of primers, polymerase chain reaction and restriction analysis. The other aspect of this dissertation is focused on the study of acyl-CoA binding protein of Cryptococcus neoformans (CnACBP) cloned for first time in our laboratory. Homologues of ACBP were found in all organisms distributed in the four kingdoms of eukaryotes, with high sequence similarity (~ 48%). Its widespread presence and their involvement in several key metabolic pathways related to the acyl-CoA ester led to the conclusion that ACBP is a housekeeping protein and not a specific protein contained a specialized cell type. Here we present an initial characterization of CnACBP that seeks to relevant issues regarding the proteins function. Our goal was to increase the still vague knowledge on how electrical charges and the presence of the binding partner may influence the structure, stability and function through thermodynamic and spectroscopic techniques. This is an initial step toward the full understanding of the role of protein in the cell.

Abstract Multifunctional enzyme type 1 (MFE-1) is a monomeric member of the hydratase/isomerase superfamily (H/I) involved in the β-oxidation of fatty acids. MFE-1 has 2-enoyl-CoA hydratase-1, Δ3-Δ2-enoyl-CoA isomerase, and several other enoyl-CoA isomerase activities at the N-terminus. The C-terminus has (3S)-hydroxyacyl-CoA dehydrogenase activity. MFE-1 can also convert certain hydroxylated C27 bile acid synthesis intermediates. In these studies, a domain assignment of MFE-1 by sequence alignment with the H/I family (domains A and B in MFE-1) and mitochondrial monofunctional 3-hydroxyacyl-CoA dehydrogenases (HAD, domains C, D and E) was proposed. This was further improved with the structural information obtained from the crystal structure of the construct containing domains B, C, D and E (MFE1-DH). The structure of MFE1-DH resembles the bilobal structure of the α-subunit of the bacterial fatty acid metabolising complex and the mammalian HAD enzyme. The N-terminal linker helix of MFE1-DH (domain B) corresponds to helix-10 of the hydratase/isomerase enzymes having residues important for substrate contacts. Domain C adopts the classical Rossmann fold and forms the first lobe of the MFE1-DH structure. The C-terminal domains D and E form the second lobe and have local symmetry between each other. This local symmetry corresponds to the D domain-mediated dimerisation of the HAD dimer. The domain deletion studies showed that the presence of domains D and E, but not domain C, was essential to obtain a functional hydratase 1 enzyme; this can be understood from stabilising contacts from domain E to the linker helix, as seen in the MFE1-DH structure. The structure of human ACBP from liver was determined with and without a physiological ligand. This structure adopts the classical four-helix bundle of the ACBP family. The ligand binding mode seen in the presence of myristoyl-CoA shows that one ligand molecule is bound jointly by the two protein molecules of the asymmetric unit such that the fatty acid tail is bound by one protein molecule, and the 3'-phosphate AMP moiety of the CoA is bound by the other protein molecule, essentially as in known complexed ACBP structures in the monomeric binding mode. The observed ligand binding mode suggests a new model for the ACBP-mediated ligand transfer observed in biochemical in vitro studies. / Tiivistelmä Tyypin 1 monitoiminen entsyymi (MFE-1) on hydrataasi/isomeraasiperheen (H/I) jäsen ja se osallistuu rasvahappojen β-oksidaatioon. MFE-1:n N-päädyssä on 2-enoyyli-CoA-hydrataasi 1- ja Δ3-Δ2-enoyyli-CoA-isomeraasiaktiivisuus sekä useita muita enoyyli-CoA-isomeraasiaktiivisuuksia. C-päädyssä on (3S)-hydroksiasyyli-CoA-dehydrogenaasiaktiivisuus. MFE-1 voi myös katalysoida tiettyjen hydroksyloitujen C27-sappihapposynteesin välituotteiden reaktioita. Tässä tutkimuksessa määritettiin MFE-1:n domeenirakenne H/I-perheen (MFE-1:n domeenit A ja B) ja 3-hydroksiasyyli-CoA-dehydrogenaasiperheen (HAD, domeenit C, D ja E) sekvenssilinjausten perusteella. Rakennetta tarkennettiin domeenit B, C, D ja E sisältävän konstruktin (MFE1-DH) kiderakenteesta saadun tiedon avulla. MFE1-DH:n kiderakenne muistuttaa bakteerien rasvahappoja hajottavan kompleksin α-alayksikön sekä nisäkkäiden HAD-entsyymin kahdesta alayksiköstä muodostuvaa rakennetta. MFE1-DH:n N-päädyn α-kierre vastaa H/I-entsyymien kierre-10:tä, jossa sijaitsee substraattikontaktien kannalta tärkeitä aminohappotähteitä. C-domeeni muodostaa Rossmann-laskoksen ja se on MFE1-DH:n rakenteen ensimmäinen alayksikkö. C-päädyssä sijaitsevat D- ja E-domeenit muodostavat yhdessä toisen alayksikön ja niiden välillä on symmetria, joka vastaa D-domeenien välittämää HAD-entsyymien dimerisaatiota. Domeenitutkimukset osoittivat, että D- ja E-domeenien läsnä olo oli välttämätöntä hydrataasi 1:n toiminnalle, mutta C-domeeni voitiin poistaa ja säilyttää hydrataasi 1 -aktiivisuus. Havainto voitiin selittää MFE1-DH:n rakenteen avulla, jossa nähdään stabiloivia vuorovaikutuksia E-domeenin ja N-päädyn α-kierteen välillä. Ihmisen maksan ACBP:n kiderakenne määritettiin fysiologisen ligandin kanssa sekä ilman ligandia. Tämä rakenne laskostuu ACBP-perheelle tyypilliseksi neljän kierteen nipuksi. Myristoyyli-CoA:n läsnä ollessa havaitussa ligandin sitoutumistavassa yksi ligandimolekyyli on sitoutunut kahden proteiinimolekyylin välille siten, että rasvahappo-osa sitoutuu toiseen proteiinimolekyyliin ja CoA:n 3'-fosfaatti-AMP-osa sitoutuu toiseen proteiinimolekyyliin kuten tunnetuissa monomeerisissä ACBP:n sitomistavoissa. Havaitun sitoutumisen avulla voidaan ehdottaa uutta mallia ACBP:n välittämälle ligandinsiirrolle, joka on havaittu aikaisemmin biokemiallisissa in vitro -tutkimuksissa.