Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Apolipoprotein E (apoE) is crucial for lipid transport and cholesterol homeostasis within the plasma and central nervous system. Binding to lipoprotein particles activates apoE allowing it to interact with cell surface receptors such as the low density lipoprotein receptor (LDLr). This mediates the clearance of apoE containing lipoproteins through an endocytosis pathway and therefore reduces plasma cholesterol level, explaining the strong anti-atherogenic effect of apoE. Nevertheless, the active conformation of apoE, critical for the receptor interaction, remains elusive. Since high resolution structure-determination methods (i.e. NMR and X-ray crystallography) are not yet applicable to large protein/lipid complexes, we are actually in need of biophysical techniques to solve the structure-activity relationship of lipidated apoE. The aim of this work was to elucidate the lipidated structure of apoE. Therefore, we reconstituted apoE/lipid particles mimicking a subspecies of high density lipoproteins (HDL) found in the plasma, starting from recombinant apoE and synthetic lipids (POPC). After optimization, a homogeneous population of reconstituted apoE/POPC lipoproteins was obtained. A thorough characterization by different biochemical methods (native PAGE, electron microscopy, FTIR…) showed that these lipoprotein particles bear 2 apoE molecules at their surface and display a discoidal shape with a diameter of 105Å. Further structural information was gathered by using a combination of lysine-directed cross-linking and mass spectrometry. We then developed a two-step modeling procedure using all the structural knowledge acquired at this point enriched with literature data to build 3D atomic resolution structural models of the apoE4/POPC complexes. First, monomeric apoE models were produced in the absence of lipids using a distance calculation program. These initial models, where apoE is folded into a helical hairpin, should reflect the conformation adopted by the protein on a disc-shaped lipoprotein. Secondly, two modeled monomeric conformations were dimerized, associated with a lipid disc, solvated and finally subjected to a molecular dynamics (MD) simulation. Our MD trajectories show an elongation of a helix in the N-terminal domain that redefines the position of Arg172, a key residue for LDLr binding, in regards to the well-defined receptor binding region (residues 136-150) of apoE. The observed elongation explains why the lipidation of apoE is an essential requirement in the activation mechanism and offers an ideal framework for the recognition of two specific LDLr ligand binding repeats needed for full binding to the LDLr. Based on an important structural change observed in our different models, we can also propose an activation mechanism relying on a regulation of the accessibility of the ligand binding region by the protein itself. Thus, the two new presented models provide important structural information at the atomic level and new insights into the mechanisms involved in apoE receptor recognition. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Background: Despite substantial research and development investment in Alzheimer's disease (AD), effective therapeutics remain elusive. Significant emerging evidence has linked cholesterol, beta-amyloid and AD, and several studies have shown a reduced risk for AD and dementia in populations treated with statins. However, while some clinical trials evaluating statins in general AD populations have been conducted, these resulted in no significant therapeutic benefit. By focusing on subgroups of the AD population, it may be possible to detect endotypes responsive to statin therapy. Methods: Here we investigate the possible protective and therapeutic effect of statins in AD through the analysis of datasets of integrated clinical trials, and prospective observational studies. Results: Re-analysis of AD patient-level data from failed clinical trials suggested by trend that use of simvastatin may slow the progression of cognitive decline, and to a greater extent in ApoE4 homozygotes. Evaluation of continual long-term use of various statins, in participants from multiple studies at baseline, revealed better cognitive performance in statin users. These findings were supported in an additional, observational cohort where the incidence of AD was significantly lower in statin users, and ApoE4/ApoE4-genotyped AD patients treated with statins showed better cognitive function over the course of 10-year follow-up. Conclusions: These results indicate that the use of statins may benefit all AD patients with potentially greater therapeutic efficacy in those homozygous for ApoE4.

Der bedeutendste Risikofaktor für die Demenz vom Azheimer Typ (DAT) ist, neben dem Alter, der Besitz des e4-Allels des Lipidtransportmoleküls Apolipoprotein E (APOE, Gen; ApoE, Protein). Neben funktionellen Unterschieden der Isoformen könnte auch die quantitative Verfügbarkeit des Proteins bei der Pathogenese der DAT eine Rolle spielen. Da ApoE als Lipidtransporter insbesondere bei plastischen Umbauprozessen in Gehirn wichtig sein könnte, wurde der Proteingehalt in Bezug auf die Schwere der DAT-assoziierten Veränderungen entsprechend der neuropathologischen Stadieneinteilung nach Braak untersucht. Neben ApoE wurde auch Apolipoprotein D (ApoD) in der Pathogenese der DAT diskutiert. Um herauszufinden, ob es hier eine Beziehung zur Schwere der neuropathologischen Veränderungen und/oder dem APOE-Polymorphismus gibt, wurde auch der ApoD-Gehalt untersucht. Bestimmt wurde der Apolipoproteingehalt in 104 nach Alter, Geschlecht, Braak-Stadium und APOE-Genotyp abgeglichenen Hippokampusproben, da diese Hirnregion im Verlauf einer Demenz früh und nachhaltig betroffen ist. Die Daten zeigen einen Anstieg von ApoE in den frühen Stadien der Erkrankung, in denen neuroplastische Reparationsvorgänge nachweisbar sind, und eine kontinuierliche Zunahme des ApoD-Gehalts mit der Schwere der neurofibrillären Veränderungen in den APOE e3/3-Proben. In den Proben, die das Risikoallel APOE e4 trugen, waren keine statistisch signifikanten Veränderungen nachweisbar. / The most important risk factor for Alzheimer´s disease (AD), besides age, is the inheritance of the e4-allele of the lipid transporting apolipoprotein E (APOE, gen; apoE, protein). Apart from functional differences of the apoE isoforms, the quantitative availability of the protein might be important in AD pathogenesis. As a lipidtransporter, apoE should be relevant especially during plastic response of neuronal structures. Hence, apoE levels were investigated in correlation to the severity of pathological changes as staged according to the neuropathological Braak classification of AD. Besides apoE, apolipoprotein D (apoD) is discussed in AD pathogenesis. To clarify whether there is a correlation to AD related neuropathological changes, apoD levels were also determined. The apolipoproteins were determined in 104 age-, gender-, Braak stage- and APOE genotype-matched samples of hippocampal protein extracts because this brain region is early and enduring affected by the disease. The data show an increase in apoE in the early stages of the disease where plastic remodelling occurs and a continuous increase in apoD with the severity of neurofibrillary changes in the APOE e3/3-group only. No statistically relevant changes were observed in samples carrying the AD-risk allele e4.

Introduction There has been much discussion on personalised medicine; however use of genotype in risk prediction for coronary heart disease (CHD) has not resulted in appreciable improvements over non-genetic risk factors. The primary aim was to determine whether candidate single nucleotide polymorphisms (SNPs) identified from genome-wide association studies improved prediction of CHD over conventional risk factors (CRF). The secondary aim was to determine whether the use of apolipoproteins or lipoprotein(a) improved risk prediction of CHD. Methods Analyses used the Edinburgh Heart Disease Prevention Study (EHDPS), with 1592 men aged 30-59 and follow-up after 20 years; and the Edinburgh Artery Study (EAS), with 1592 men and women aged 54-75 and 15 years of follow-up. Candidate SNPs were identified by systematic literature reviews. CHD status was evaluated as severe (myocardial infarction or coronary revascularisation), and any (severe CHD, angina or non-specified ischaemic heart disease). Cox proportional hazards models were used to evaluate addition of candidate SNPs or lipids to models containing CRF. Results A group of genome-wide significant SNPs resulted in a non-significant improvement in C-index for severe CHD (0.038, p=0.082), and a significant improvement in C-index for any CHD (0.042, p=0.016); the associated net reclassification improvements (NRI) were 20.5% and 18.7%, respectively. Regression trees identified SNPs that were predictive of the remaining variance after adjusting for CRF; this resulted in a significant improvement in C-index for any CHD (0.031, p=0.008). The NRI were 11.0% and 9.6% for severe and any CHD, respectively. When compared with HDL cholesterol/total cholesterol, apolipoprotein AI/total cholesterol yielded a NRI of 3.3% for severe CHD. Lipoprotein(a) improved prediction of severe CHD, with a non-significant improvement in C-index (0.020, p=0.087), and NRI of 11.8%. Conclusion The results of this study indicate that a well selected group of candidate SNPs can improve risk prediction for CHD over-and-above CRF. The inclusion of lipoprotein(a), along with CRF, appeared to improve prediction of severe CHD, but not any CHD.

Les apolipoprotéines L (APOL) forment une famille de protéines conservées chez les mammifères. L’APOL6 murine est principalement exprimée par les adipocytes présents dans le tissu adipeux. Dans un modèle de culture d’adipocytes, l’adipogénèse a causé l’induction de l’expression de l’APOL6. Celle-ci a pu encore être modulée à la hausse par de l’IFNγ, et à la baisse par du TGFβ. Des facteurs élevant la concentration en AMP cyclique ont aussi permis de diminuer l’expression d’APOL6. In vivo, lorsque des souris APOL6 KO ont été nourries par un régime riche en graisses, elles ont pris moins de poids que les souris WT correspondantes. De plus, les adipocytes des souris APOL6 KO obèses étaient plus petits que ceux des contrôles WT. Finalement, la recherche de protéines interagissant avec l’APOL6 par immunoprécipitation a permis de mettre en évidence une majorité de protéines associées au cytosquelette d’actine. En conclusion, l’APOL6 semble être associée au cytosquelette d’actine des adipocytes et permettrait la régulation de la taille de leurs gouttelettes lipidiques. / Apolipoproteins L (APOL) are a family of conserved proteins among mammals. Murine APOL6 is mainly expressed by adipocytes in the adipose tissue. In a model of in vitro adipocyte cell culture, adipogenesis induced the expression of APOL6. This expression increased with IFNγ and decreased with TGFβ. Cyclic-AMP elevating agents also decreased the expression of APOL6. In vivo, APOL6 KO mice that were fed with a high fat diet gained less weight than their wild type (WT) counterparts. Furthermore, adipocytes from obese APOL6 KO mice were smaller than those from WT controls. Finally, immunoprecipitation experiments showed that APOL6 probably interacted with actin cytoskeleton proteins within adipocytes. In conclusion, APOL6 is likely associated with the actin cytoskeleton in adipocytes and could be involved in the regulation of the size of lipid droplets. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. It is a non-covalent, homodimeric and N-glycosylated protein, which is regulated in a tissue-specific manner and is dependent on an activator protein, apolipoprotein CII. Dissociation of active LPL dimers to monomers leads to loss of activity. This was previously found to be an important event in the rapid regulation of LPL in tissues. The mechanisms involved in the processing of LPL to active dimers, as well as in LPL inactivation through monomerization, were unknown. We have investigated the folding properties of the LPL protein, in particular the requirements for LPL to attain its active quaternary structure and to remain in the native conformation. On expression of LPL in insect cells we found that most of the LPL protein was synthesized in an inactive monomeric form. By co-expression of LPL with human molecular haperones, especially with calreticulin (CRT), the activity of LPL increased greatly, both in the cells and in the media. The effect of CRT on LPL activity was not due to increased levels of the LPL protein, but was due to an increased proportion of active dimeric LPL. Co-immunoprecipitation experiments showed direct interaction between LPL and CRT supporting the idea that this ER-based molecular chaperone supports the formation of active LPL dimers. We showed that, bis-ANS, the aromatic hydrophobic probe 1,1.-bis(aniline)-4,4.- bis(naphthalene)-8,8.disulfonate, can be used to obtain specific information about the interaction of LPL with lipid substrates and with apoCII. Bis-Ans was found to be a potent inhibitor of LPL activity, but apoCII prevented the inhibition. Our results suggest that bis-Ans binds to three exposed hydrophobic sites, of which one is at or close to the binding site(s) for apoCII. In studies of the mechanisms responsible for the spontaneous inactivation of LPL, we showed that active LPL is a dynamic dimer in which the subunits rapidly exchange partners. The rapid equilibrium between dimers and monomers exists even under conditions where LPL is relatively stable. This supports the idea that the dimer is in equilibrium with dimerization-competent, possibly active monomers. This dimerization-competent intermediate was also implicated in studies of the inactivation kinetics. The inactive LPL monomer was found to have a stable, defined conformation irrespective of how it was formed. The main differences in conformation between the inactive monomer and the active dimer were located in the middle part of the LPL subunit. Experiments with bis-Ans demonstrated that more hydrophobic regions were exposed in the inactive monomer, indicating a molten globule conformation. We concluded that the middle part of the LPL subunit is most likely engaged in the formation of the active LPL dimer. The dimerization-competent LPL monomer is a hypothetical conformational state, because it has not been possible to isolate it. To study complete refolding of LPL we used fully denatured LPL and were able to demonstrate that the recovery of LPL activity was about 40% when the denaturant was diluted by a buffer containing 20% human serum and 2M NaCl. Further studies identified calcium as the component in serum that was crucial for the reactivation of LPL. The refolding of LPL was shown to involve at least two steps, of which the first one was rapid and resulted in folded, but inactive monomers. The second step, from inactive monomers to active dimers, was slow and calcium-dependent. Also inactive monomers isolated from human tissue were able to recover activity under the influence of calcium. We proposed that calcium-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity. In conclusion, LPL is a relatively unstable enzyme under physiological conditions due to its noncovalent dimeric structure. The energy barrier for folding to the active dimer is high and requires the presence of calcium ions and molecular chaperones to be overcome. The dimeric arrangement is probably essential to accomplish rapid down-regulation of LPL activity according to metabolic demand, e.g. in adipose tissue on fasting.

Apolipoprotein (apo) A-V is the most recently discovered member of a protein family responsible for the structure and metabolic fate of plasma lipoproteins. While most of the apolipoproteins are well characterized with regard to structure, interactions and function, the role of apoA-V is not well understood. ApoA-V is synthesized only in liver and is present in blood at much lower concentration than the other apolipoproteins. Although apoA-V is firmly established as an important determinant for plasma triacylglycerol (TG) metabolism, the mechanism is unclear. ApoA-V has been suggested to act through 1) an intracellular mechanism affecting lipoprotein assembly and secretion, 2) direct or indirect activation of lipoprotein lipase (LPL), or 3) interaction with endocytotic lipoprotein receptors. Two other novel players involved in the clearance of lipoproteins are angiopoietin-like protein (ANGPTL) 3 and 4. Previous studies have shown that the coiled-coil domain (ccd) of ANGPTL3 and -4 can inactivate lipoprotein lipase (LPL). The functional site of action of LPL is at the capillary endothelium, but the enzyme is synthesized mostly in adipocytes and myocytes and has to be transported by trancytosis to the luminal side of endothelial cells. Both ANGPTLs are present in tissues and in the circulating blood, but it is not known were the inactivation of LPL normally takes place. The aim of this thesis was to investigate the mechanism by which apoA-V exerts its effect on TG metabolism and to investigate in further detail how ANGPTLs act on the LPL system. Binding of apoA-V to receptors involved in lipoprotein metabolism was investigated by surface plasmon resonance technique (SPR). ApoA-V was found to bind to the LDL receptor related protein 1 (LRP1) and to the mosaic type 1 receptor sorLA. Binding could be competed by receptor associated protein (RAP) or by heparin, and was calcium dependent. We concluded that apoA-V binds to the LA-repeats of these receptors. In further experiments apoA-V was shown to increase binding of TG-rich chylomicrons to the receptors. This demonstrated a possible mechanism for the TG-lowering effect of apoA-V in vivo. A putative binding region in apoA-V for heparin and receptors was investigated by site-directed mutagenesis. Two positively charged amino acid residues were changed (Arg210Glu/Lys211Gln), resulting in decreased binding to heparin and to LRP1 and thus the localization of one important functional region in apoA-V. Since the receptor sorLA also contains a Vsp10p domain, another Vsp10p domain family member, sortilin, was investigated. ApoA-V was found to interact also with this receptor. In experiments with human embryonic kidney cells transfected with sorLA or sortilin, apoA-V was found to bind to cell surfaces and to be rapidly internalized while co-localized with the receptors on the way to lysosomes for degradation. Additional apoA-V mutants, identified in patients with severe hypertriglyceridemia, were investigated with regard to effects in vitro on LPL activity and receptor binding. The most severe mutants displayed null binding to LRP1, whereas the effect on LPL activity was retained. These results suggest that lack of receptor interaction mirrors the loss of biological function in a better way than the in vitro effect on LPL activity. We noted that ccd-ANGPTL3 and -4 did not prevent the LPL-mediated uptake of chylomicron-like lipoproteins in primary murine hepatocytes. Therefore LPL activity was measured after pre-incubation with ccd-ANGPTL3 or 4 in the presence or absence of TG-rich lipoproteins. Physiological concentrations of lipoproteins were found to protect LPL from inactivation by ccd-ANGPTLs. Investigation by SPR demonstrated that the ccd-ANGPTLs did not bind to the lipoproteins. Other experiments showed that less than 1% of ANGPTL4 in human serum was bound to TG-rich lipoproteins. This implies that the known binding of LPL to TG-rich lipoproteins stabilizes the enzyme and protects it from inactivation by ANGPTLs. We conclude that the normal levels of ANGPTLs in plasma are too low to affect the LPL-system and that inactivation of the enzyme by ANGPTLs is more likely to occur locally in the extracellular interstitium of tissues where LPL is en route to its endothelial binding sites and where the concentrations of the TG-rich lipoproteins are low.