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Identifier 000430934
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Title The role of proteolytic systems in the control of lipid metabolism
Alternative Title O ρόλος πρωτεολυτικών μηχανισμών στον μεταβολισμό λιπιδίων
Author Γεωργιλά, Κωνσταντίνα
Thesis advisor Ηλιόπουλος, Αριστείδης
Abstract Apolipoprotein A-I (ApoA-I) is involved in reverse cholesterol transport as a major component of HDL, but also conveys anti-thrombotic, anti-oxidative, anti-inflammatory and immuneregulatory properties that are pertinent to its protective roles in cardiovascular, inflammatory and malignant pathologies. The aim of this PhD Thesis is to define post-transcriptional mechanisms responsible for the regulation of intracellular ApoA-I levels and decipher putative intracellular functions of ApoA-I, questions which remain poorly explored. Data presented in chapter 1 address the role of proteolytic pathways, autophagy and the ubiquitin-proteasome system (UPS) in ApoA-I regulation in hepatocytes and hepatoma cells as in vitro models to study the impact of genetic and chemical inhibitors of autophagy and the proteasome on ApoA-I by immunoblot, immunofluorescence and electron microscopy. Different growth conditions were implemented in conjunction with mTOR inhibitors to model the influence of nutrient scarcity versus sufficiency on ApoA-I regulation. Hepatic ApoA-I expression was also evaluated in high fat diet-fed mice displaying blockade in autophagy. The results documented that under nutrient-rich conditions, basal ApoA-I levels are sustained by the balancing act of autophagy and of mTORC1-dependent de novo protein synthesis. Pharmacologic inhibition of autophagy and not the proteasome resulted in the accumulation of ApoA-I in hepatocytes. To dissect the molecular pathway that governs ApoA-I degradation by autophagy we used genetic silencing of key modulator of autophagic machinery and monitored ApoA-I levels. We found that ApoA-I proteolysis occurs via a canonical autophagic pathway involving Beclin1 and ULK1 and the receptor protein p62/SQSTM1 that targets ApoA-I to autophagosomes. We next analyzed ApoA-I levels, under conditions of starvation that induce autophagy and in parallel block anabolic processes as translation through mTOR pathway. We found that upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis. Collectively, the data presented in chapter 1 underscore the major contribution of posttranscriptional mechanisms to ApoA-I levels which differentially involve mTORC1-dependent signaling to protein synthesis and autophagy, depending on nutrient availability. Given the established role of ApoA-I in HDL-mediated reverse cholesterol transport, this mode of ApoA-I regulation may reflect a hepatocellular response to the organismal requirement for maintenance of cholesterol and lipid reserves under conditions of nutrient scarcity. As hepatic expression of ApoA-I responds to nutrient availability, we were interested to explore whether ApoA-I may act as a mediator for specific cellular energetic requirements and as modulator of intracellular lipid metabolism under conditions of autophagic blockade and/or increased lipid load. Data presented in chapter 2 delineate the putative intracellular function of ApoA-I in the regulation of lipid metabolism. Intrigued by published evidence that, (1) suppression of autophagy leads to accumulation of large size lipid droplet (LD) in vitro and steatosis in vivo; (2) ApoA-I ablation in HFD-fed mice results in steatosis; and (3) ApoA-I accumulates in response to inhibition of autophagy, we hypothesized that intracellular ApoA-I may physiologically function to suppress lipid and cholesterol overload under conditions of autophagy blockade. Indeed, the knock-down of ApoA1 in the hepatoma cell line HepG2 resulted in the accumulation of large size lipid droplets, an effect that was amplified upon simultaneous inhibition of autophagy. This ApoA-I function was independent of autophagy, as ApoA-I depletion did not significantly affect autophagic flux. To provide mechanistic insights into the modulation of intracellular lipid stores by ApoA-I, we used a hypothesis-free approach that entails the unbiased interrogation of the transcriptome of ApoA-I depleted HepG2 cells. We identified enrichment of biological processes linked to lipid and cholesterol metabolism and an association with fatty liver and metabolic diseases. Interestingly, a significant number of genes related to cholesterol and lipid biosynthesis were commonly up-regulated by autophagy blockade and ApoA-I depletion which, when combined, led to a marked increase in metabolic gene expression compared to either treatment alone. These observations align with the LD accumulation ensued by these treatments and indicate that intracellular ApoA-I and autophagy converge to transcriptionally control de novo lipid and cholesterol biosynthesis. The significance of these findings in human disease is highlighted by our mining of hepatocellular carcinoma RNAseq data which confirmed that ApoA1 expression levels are inversely correlated with genes implicates in lipid and cholesterol biosynthesis, such as HMGCR, HMGCS, FASN, LPIN1 and ACACA. Expression analysis of key transcriptional modulators of lipid and cholesterol biosynthetic genes upon ApoA1 knock-down and/or autophagic inhibition revealed that inhibition of autophagy affects SREBP2 maturation but not FOXO1/3, whereas the RNAi-mediated depletion of ApoA1 leads to marked phosphorylation of FOXO1/3 but does not impact SREBP2 cleavage which is required to generate the transcriptionally active SREBP2. These findings align both with the impact of ApoA1 depletion and autophagy inhibition on inducing the expression of a common set of cholesterol and lipid biosynthesis genes and their amplifying effects on gene expression when combined. As FOXO1/3 phosphorylation is mediated by Akt activation, the effect of ApoA-I depletion on the Akt signaling pathway was examined. It was found that the knock-down of ApoA1 leads to mTORC2-dependent phosphorylation of Akt at Ser473 and that suppression of the mTORC2/Akt axis diminishes the effect of ApoA-I depletion on HMGCR and HMGCS1 expression. Our preliminary data demonstrate that ApoA-I physically interacts with both Akt1 and Akt2 at least when overexpressed in HEK293T cells. Further studies exploring the mechanism by which ApoA-I physiologically functions to moderate Akt activation are warranted. Beyond the upregulation of lipid and cholesterol biosynthesis processes, the transcriptome of dually ApoA-I depleted and autophagy-inhibited HepG2 cells is enriched in downregulated genes related to cell cycle control and an overall association with neoplastic diseases. We therefore hypothesize that the reduction in ApoA-I coupled with autophagy blockade, could provide a cellular environment of lipid overload with a pro-survival potential that could drive malignant phenotypes in the liver. In line with this notion, the knock-down of ApoA1 in HepG2 cells protects against lipotoxicity and genotoxicity, and in parallel provides ER-stress resistance as indicated by the transcriptional suppression of genes involved in lipid overload-mediated ER stress, facilitating cell survival. Finally, our preliminary in vivo data, in accordance with our in vitro results, demonstrate that ApoA-I ablation in mice with liver specific ablation of ATG5 exaggerates steatosis and carcinogenic phenotypes. Collectively, our study sheds light into the regulation of hepatic ApoA-I by defining a major post-transcriptional pathway responsible for the control of intracellular ApoA-I levels. Under nutrient-rich conditions, ApoA-I expression is sustained by the balancing acts of basal autophagy and of mTORC1-dependent de novo protein synthesis. In contrast, upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis. In addition, we unveil a novel role for ApoA-I in hepatic lipid homeostasis. ApoA-I functions as a break to lipid accumulation which is particularly evident under conditions of autophagic inhibition. We also found that autophagy blockade, besides its direct involvement in the regulation of intracellular lipid stores through lipophagy and modulation of lipolytic mechanisms, acts in the transcriptional regulation of cholesterol and fatty acid biosynthetic gene expression by affecting SREBP2 maturation. The effect of autophagy inhibition on gene expression is augmented by ApoA1 knock-down which induces mTORC2-dependent Akt phosphorylation inactivating the transcription factor FOXO1/3, a negative regulator of lipid and cholesterol biosynthesis genes. The novel intracellular functions of ApoA-I identified in this PhD Thesis are likely to transform our understanding of lipid and cholesterol metabolism and expand our understanding of the in vivo physiological roles of ApoA-I. The identification of ApoA-I as a novel putative Akt-interacting protein, once consolidated with additional experiments, will also expand our knowledge of this key kinase in different tissues. Results in this PhD Thesis also demonstrate that reduced levels of ApoA-I under conditions of autophagy inhibition, as observed in hepatocellular carcinoma, are associated with amplified lipid and cholesterol biosynthetic gene expression, lipid droplet accumulation, enhanced survival in vitro and exaggerated hepatocarcinogenesis in vivo. Future studies will aim at dissection of the mechanism of ApoA-I modulation of Akt phosphorylation and provide further insights into the implication of ApoA-I regulation in liver pathologies associated with increased lipid load and carcinogenesis.
Language English
Subject Apolipoprotein A 1
Autophage
Απολιποπρωτεϊνη Α 1
Αυτοφαγία
Λιπιδικός μεταβολισμός
Issue date 2020-08-05
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/d/7/c/metadata-dlib-1596699443-151177-26152.tkl Bookmark and Share
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