A LDL contitui-se de uma fase lipídica, uma esfera composta principalmente de um núcleo de colesterol esterificado envolto por uma camada de fosfolipídeos. A fase lipídica junta-se uma única molécula protéica, a polipoproteina B (apo B). A LDL é removida da circulação por mediação de receptores específicos, os receptores B. E, que reconhecem não só a apo B da LDL mas também a apo E, que está presente em outras lipoproteínas. Estudou-se neste trabalho a influencia da apo B no metabolismo da LDL, através de um modelo de emulsões semelhantes à fase lipídica da LDL, constituídas de oleato de colesterol (29%), fosfatidilcolina (69%) e trioleina (2%), preparadas por sonicação, seguida de ultracentrifugação. As emulsões, com seus componentes lipídicos marcados isotopicamente, foram associadas à apo B e injetadas no rato, determinando-se sua remoção plasmática e captação pelos diversos órgãos do animal. Os resultados foram comparados com o metabolismo da emulsão sem associação da apo B a sua estrutura e com o da LDL natural. Verificou-se que a presença de apo B retardou a remoção plasmática das partículas da emulsão. Por outro lado, o oleato de colesterol da emulsão com apo B foi removido do compartimento plasmático numa taxa semelhante à da LDL natural, o que demonstra a adequação do modelo utilizado à situação fisiológica. Tanto as emulsões quanto a LDL natural foram captadas principalmente pelo fígado. Entretanto, a captação hepática da emulsão sem apo B foi maior a da emulsão com apo B e a da LDL. A incubação das emulsões com HDL do plasma de rato mostrou que a emulsão sem apo B adquiriu mais apo E que a emulsão com apo B. Portanto, essas diferenças de comportamento metabólico provavelmente se devem à maior afinidade dos receptores B, E hepáticos por partículas que contem mais apo E, em comparação às que contêm a apo B. O aumento da atividade dos receptores B, E, induzido pelo tratamento com 17 α-etinil estradiol, resultou no aumento da cinética plasmática das duas emulsões. Porém, à taxa de remoção plasmática da emulsão sem apo B também foi maior no grupo tratado com o estrógeno. Esses dados indicam que as emulsões com apo B associada a sua estrutura apresentam comportamento metabólico muito semelhante ao da LDL. Portanto, o modelo das emulsões análogas à LDL parece ser um instrumento eficiente no estudo do metabolismo daquela lipoproteína. / The effects of apolipoprotein B (apo B) on the metabolism of emulsions constituted of cholesteryl oleate (29%), phosphatidycholine (69%) and triolein (2%) were studied in rats. After intra-arterial injection of the radiolabelled emulsions, plasma removal of the emulsions was reduced in presence of apo B. On the other hand, the cholesteryl ester moiety of the apob B emulsion was removed at the same rate as native LDL. Emulsions and LDL were taken up mainly by the liver 24 h after the injection. However, the hepatic uptake of the apo B emulsion was similar to LDL and lower than that of the apo B-free emulsion. These differences in metabolic behaviour were probably due to the lower hepatic B, E, receptor affinity to apo B contained in the emulsions associated to apo B and in LDL, compared to the apo B-free emulsions. The latter, as confirmed in the in vitro experiments, is capable of adsorbing more apo E, and this apolipoprotein has higher affinity for the receptor. Enhanced receptor activity induced by pre-treatment of the rats with 17 α-ehymylestradiol resulted in augmented plasma removal of both emulsions but nonetheless the cholesteryl ester plasma removal of the apo B-containing particles was still lower, compared to that of the apo B-free emulsion. These data indicate that apo B-containing emulsion exhibits metabolic behaviour similar to that of LDL, and this emulsion can be an adequate tool to test LDL metabolism.

Apolipoprotéines L est une famille nouvellement caractérisée en humain sans une fonction patho- physiologique définitive. Ces protéines sont classiquement considérées être impliquées dans le transport et métabolisme des lipides, principalement due à l'association de son premier membre de la famille sécrétée l’apolipoprotéine L1 aux particules des lipoprotéines de haute densité. Néanmoins, le reste des membres sont des protéines intracellulaires (absence de domaine de peptide signal). Apolipoprotéine L1 a été initialement identifiée comme l'élément clé du facteur trypanolytique dans le sérum humain. L'exploration de la séquence des différents apolipoprotéines L a révélé un domaine distinct «B cell lymphoma-2 homology domain 3» ayant des similitudes structurelles et fonctionnelles avec le domaine B cell lymphoma-2 homology domain 3 des protéines de la famille B cell lymphoma-2. Ainsi la découverte de ce domaine peut contribuer à la compréhension de la fonction et rôle des apoLs dans différents mécanismes et processus tels que la mort cellulaire programmée, la prolifération cellulaire, le métabolisme cellulaire .Notre étude visait à caractériser les fonctions de patho- physiologique du premier membre de la famille «apolipoprotéine L1 ». L’expression de l’apolipoprotéine L1 ARNm, à partir de 48 carcinomes papillaires de la thyroïde, a été évaluée par des études à haut débit et normalisée à un pool de tissus normal de la thyroïde. Une confirmation de PCR en temps réel valide ainsi la surexpression d’apoL1 dans 91,67 % des cas testés. Le niveau élevé de l’apolipoprotéine L1 ARNm est en corrélation avec une expression protéique élevée dans les échantillons histologiques (70%), et détermine que les cellules folliculaires de la thyroïde dans la zone de la tumeur sont les cellules principales responsables de l’expression spécifique de l’apolipoprotéine L1. Nous avons étudié l'expression apolipoprotéine L1 dans le modèle de cancer pour approfondir notre compréhension des relations reliant cette expression distincte dans le cancer papillaire de la thyroïde et son rôle et fonction concernant le métabolisme du cancer (de reprogrammation métabolique :effet Warburg).7En outre, la localisation de l’apolipoprotéine L1 dans la mitochondrie des cellules cancéreuses de la thyroïde ainsi que dans la mitochondrie de levure, a été le point de départ de la recherche dans ce nouveau modèle, il nous a permis de révéler et d'introduire de nouvelles hypothèses pour expliquer l'effet inhibiteur de l’apolipoprotéine L1 en fonction des conditions métabolique variantes et l’effet pléotropiques de l’apolipoprotéine L1 sur la levure (dommages des mitochondries et vacuoles). Dans ce manuscrit, nous avons décrit nos efforts à mettre en évidence la spécificité d'expression de l’apolipoprotéine L1 dans le cancer papillaire thyroïdien notamment au niveau de la transcription ainsi que la localisation mitochondriale et l'interférence probable avec les voies métaboliques. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L’entrée du virus de l’hépatite C (VHC) dans les hépatocytes est un événement multi-étapes complexe, impliquant un certain nombre de facteurs cellulaires. Elle est initiée par la fixation des particules virales sur des structures d’héparanes sulfates (HS) présentes à la surface de l’hépatocyte. Cette étape initiale reste cependant peu comprise. En effet, en raison de l’interaction de la particule virale du VHC avec des lipoprotéines, la contribution exacte des différents composants du virion à cette interaction reste controversée. Au cours de cette thèse, nous avons étudié le rôle potentiel de protéines d'enveloppe du VHC et de l'apolipoprotéine E dans l'étape de liaison aux HS. Nous avons d’abord montré que la délétion de la région hypervariable 1 (HVR1), une région précédemment proposée pour participer à l’interaction avec les HS, n'avait aucun effet sur la liaison du virion aux HS, indiquant que cette région n'est pas impliquée dans cette interaction. Nous avons également utilisé des anticorps monoclonaux neutralisants reconnaissant différentes régions des glycoprotéines d'enveloppe du VHC dans un test de compétition utilisant des billes d’agarose couplées à l’héparine, un homologue structural des HS, pour précipiter le virus. Bien que les glycoprotéines d’enveloppe du VHC dissociées de la particule virale interagissaient avec l'héparine, aucun de ces anticorps n’était capable d'interférer avec l'interaction entre la particule virale et l’héparine, suggérant fortement que les glycoprotéines d'enveloppe du VHC présente à la surface des virions ne sont pas accessibles pour interagir avec les HS. En revanche, nos résultats d’études cinétiques, d’interaction avec l’héparine ainsi que les expériences d'inhibition avec des anticorps anti-apolipoprotéine E indiquent que cette apolipoprotéine joue un rôle majeur dans l'interaction initiale entre le VHC et les HS. Enfin, la caractérisation des déterminants structuraux des HS nécessaires à l'infection par le VHC, à l’aide d’ARN interférents ciblant des enzymes impliquées dans la voie de biosynthèse des HS et par compétition avec des héparines modifiées, indique que la N-sulfatation et la 6-O-sulfatation sont nécessaires pour l’initiation de l'infection par le VHC. Par contre la 2-O-sulfatation n’est pas indispensable pour l’étape d’entrée cellulaire du VHC. Enfin, nous avons également montré que la taille minimale des oligosaccharides d’HS requise pour l'infection par le VHC est un decasaccharide. En conclusion, l’ensemble de ces données indique que le VHC détourne l'apolipoprotéine E pour initier son interaction avec des structures d’HS spécifiques. / Hepatitis C virus (HCV) entry into hepatocytes is a complex multistep process involving a series of cellular factors. HCV entry is initiated by the binding of viral particles to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS-binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction. Neutralizing monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pull-down assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with virion-heparin interaction, strongly suggesting that, at the virion surface HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pull-down and inhibition experiments with anti-apolipoprotein E antibodies indicate that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of HS structural determinants required for HCV infection by silencing enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation are required for HCV infection, and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. The enzyme catalyzes hydrolysis of triacylglycerols (TG) of chylomicrons and of very low density lipoproteins (VLDL). However, the mechanisms involved in the regulation of this protein are not fully understood yet. Therefore, the aim of the theses is to study selected aspects of LPL activity regulation. Recently discovered apolipoprotein A-V (apo A-V) substantially affects triglyceridemia and it is presumed that it may function as LPL activator. However, its concentration in the blood is extremely low and we therefore investigated whether most of apo A-V could be bound to the heparan sulfate proteoglycan (HSPG) of vascular wall similarly to LPL. Intravenous heparin application in healthy volunteers resulted in an expected increase in LPL activity but apo A-V concentration did not change. Our results do not support the hypothesis that most of apo A-V is bound to HSPG of the capillary endothelium. An alcohol consumption plays also a role in LPL regulation - the long-term moderate alcohol consumption is known to increase enzyme activity; on the contrary, it is presumed that LPL activity is inhibited immediately after alcohol consumption. However, the direct evidence for such a premise is missing. The other aim of the theses was to...

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Agrégation de l'enseignement supérieur, Orientation sciences / info:eu-repo/semantics/nonPublished

High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in arteries contributing to atherosclerosis. In hereditary amyloidosis (AApoAI), a potentially fatal disease, N-terminal fragments of variant apoA-I deposit in vital organs and damage them. There is no cure for apoA-I amyloidosis and its structural basis is unknown. Previously, AApoAI mutations were mapped on the crystal structure of the human C-terminally truncated Δ(185-243)apoA-I. The results suggested that the mutation-induced destabilization of the lipid-free protein initiates β-aggregation. Our biophysical studies showed that amyloidogenic mutations G26R, W50R, F71Y and L170P did not necessarily destabilize the native structure, prompting us to search for additional triggers of apoA-I misfolding. We mapped residue segments predicted to promote β-aggregation (termed amyloid hot spots) on the atomic structure of ∆(185-243)apoA-I. The results suggested that perturbed packing of these hot spots, particularly residues 14-22, triggers amyloidosis. This enabled us to propose the first molecular mechanism of apoA-I misfolding. To explore a potential mechanism, we combined structural, stability, dynamics and functional studies of several amyloidogenic mutants and a non-amyloidogenic control, L159R. All mutants reduced structural protection of the segment 14-22, supporting our hypothesis that increased dynamics of this segment triggers AApoAI. The non-amyloidogenic mutant showed helical unfolding near the mutation site indicating susceptibility to proteolysis. We propose that the major factors that make apoA-I amyloidogenic are reduced protection of the major amyloidogenic segments combined with the structural integrity of the four-helix bundle to facilitate protein aggregation. Together, our results suggest that the fate of apoA-I in vivo depends on the balance between its misfolding, proteolysis, and protective protein-lipid interactions. Our structural and bioinformatics analysis of other members of the apolipoprotein family (A-II, A-IV, A-V, B, C-I, C-II, C-III, E, SAA) showed that apolipoproteins’ propensity to form amyloid is rooted in the proteins’ hydrophobicity, which is key to the lipid binding ability. The overlap of functional and pathologic interfaces suggests competition between normal protein function and misfolding. Therefore, increasing apolipoprotein retention on the lipid surface provides a potential therapeutic strategy against amyloidosis.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The incidence of Metabolic Syndrome (MetS) is increasing worldwide and accompanied by elevated risks for cardiovascular disease (CVD) and other subsequent comorbidities. MetS is associated with increased circulating triglycerides. A key enzyme involved in triglyceride (TG) clearance is lipoprotein lipase (LPL) whose activity is modulated by a variety of factors. Recent literature has identified the importance of angiopoietin-like proteins (ANGPTL) as regulators of LPL activity and has hypothesized a model in which three of these proteins interact with LPL to regulate the partitioning of TG metabolism from adipose to skeletal muscle. The work detailed in this dissertation adds to the model of ANGPTL regulation of LPL by establishing how ANGPTL8 modulates the ability of ANGPTL3 and ANGPTL4 to inhibit LPL activity in the bloodstream and localized environments, respectively. In the updated model, elevated insulin concentrations result in increased hepatic ANGPTL3/8 secretion and increased ANGPTL4/8 in adipose tissue. ANGPTL3/8 works as an endocrine molecule to inhibit skeletal muscle LPL from hydrolyzing circulating TG. Simultaneously, ANGPTL4/8 works in a paracrine mechanism to bind LPL on the endothelial vasculature adjacent to adipose tissue to alleviate ANGPTL4-mediated LPL inhibition and also prevent ANGPTL3/8 inhibition of localized LPL. Thus, in the postprandial state free fatty acids (FFA) from the hydrolysis of TG are directed into adipocytes for storage. Under fasting conditions, ANGPTL8 production is decreased in adipocytes and hepatocytes. This decreased production results in diminished ANGPTL4/8 and ANGPTL3/8 secretion from their respective tissues. As a result, ANGPTL4 inhibits adipocyte localized LPL activity while ANGPTL3 at physiological concentrations has minimal effect on LPL activity. Furthermore, any ANGPTL3/8 which is produced has its LPL-inhibitory ability diminished by the circulating apolipoprotein ApoA5. LPL is more active in skeletal muscle compared to adipose tissue where energy is shunted towards utilization in the muscle and away from storage in adipose tissue. A complete understanding of LPL regulation by ANGPTL proteins can potentially provide therapeutics targets for MetS.