Cholestérol

Résumé

Le cholestérol est un lipide de la famille des stérols. Sa molécule, plate et rigide, est un constituant essentiel de la membrane des cellules de presque tous les animaux, contrôlant ses propriétés physiques (rigidité, courbure, perméabilité). Le cholestérol joue un rôle central dans de nombreux processus biochimiques. Le mot cholestérol désigne une molécule unique. Les termes « bon cholestérol » (HDL) et « mauvais cholestérol » (LDL) désignent en réalité deux types de transporteurs du cholestérol dans le sang, les lipoprotéines de haute densité et les lipoprotéines de basse densité (voir notamment la teneur en cholestérol dans l'alimentation et l'athérosclérose). Histoire et étymologie Le cholestérol est découvert en 1758 par le chimiste français François Poulletier de La Salle, sous forme solide dans les calculs biliaires. Il est d'abord nommé cholestérine en 1814 par le chimiste français Michel-Eugène Chevreul, du grec (« bile ») et (« solide »). En 1929 il est reno

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Mitochondrial respiratory chain dysfunction disrupts intracellular cholesterol homeostasis

Christopher Tadhg James Wall

Mitochondrial diseases are rare and severe conditions with debilitating symptoms. Biochemical defects in mitochondria however are common. The difference between these two frequencies is suspected to lie in the capability of the cells to adapt to the homeostatic disbalance. Cells activate retrograde signalling pathways in response to mitochondrial dysfunction, activating cellular transcriptional programs to correct for the disbalance. The full extent of the retrograde signals that are induced by mitochondrial dysfunctions still remains unknown. Studying these retrograde signalling pathways can lead to insights how to make the difference between restoring the homeostasis and a detrimental disease. Investigating the transcriptional responses to artificially induced mitochondrial dysfunction has proven to be lucrative in unveiling novel responses to mitochondrial defects. In this work we studied the transcriptional responses to three chemical models of mitochondrial respiratory chain dysfunction. In line with recently published work, we found a commonly upregulated stress response in all models. Interestingly, shared between all the models was a downregulation of genes enriched in cholesterol biosynthesis pathways. Upon closer examination this encompassed, almost without exception, all transcripts of the mevalonate pathway. This coordinated downregulation of transcripts had functional consequences on the output of the pathway, lowering the abundance of cholesterol precursor metabolites. Other metabolites in more upstream regions of the pathway were changed differently depending on the complex affected, suggesting other regulatory influences involved. Upon closer mechanistic investigation to the cause of the transcriptional suppression we found the main transcription factor responsible for mevalonate pathway gene transcription, the Sterol Response Element Binding Protein 2 (SREBP2), to be inhibited during complex I impairment. This was most evident in sterol depleted conditions where SREBP2 did not activate to normal levels. A decreased activation of SREBP2 is signature for an elevated concentration of ER-cholesterol. Indeed, other ER-bound cholesterol sensitive proteins confirmed this, showing a similar decreased abundance following complex I impairment in both normal and low lipid conditions. To investigate the source of this cholesterol we measured various cholesterol levels. No changes in the levels of total cholesterol (free cholesterol plus esterified cholesterol) were detected. Also, the fraction of free cholesterol was not significantly affected by complex I inhibition. Using the fluorescent cholesterol stain filipin, we were able to measure a modest but reproducible increase in intracellular free cholesterol. This provided novel mechanistic insight into the inhibition of ER sterol sensors and mevalonate pathway activity by mitochondrial dysfunction. Several well-known retrograde signalling pathways AMPK, ISR and mTOR did not explain the changes observed. The exact mechanism through which mitochondria incite an increase in intracellular free cholesterol remains unknown. Taken together this work can provide a mechanistic link between mitochondrial function and intracellular cholesterol homeostasis and could possibly link mitochondrial function to circulating lipoprotein homeostasis. Further work should focus on elucidating the possible mechanism, where differences in complex functions could provide a hint in the right direction.

EPFL2022

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Water is one of the most abundant molecules in the universe. It forms a hydrogen bond network with unique structure and dynamics. Hydrogen bonding is highly relevant to many biological processes including membrane formation, protein folding, adsorption, and lubrication. However, the underlying molecular interactions within water and between water and membranes are difficult to study and hence not fully understood. In this thesis, we utilize second harmonic scattering (SHS) to investigate inhomogeneities in bulk water, probe structural changes in lipid bilayer membranes, and detect and characterize protein-membrane interactions. Firstly, we explore the structure of bulk water in comparison to a common model liquid, carbon tetrachloride. Combining SHS measurements and molecular dynamics simulations, we report on the nanoscale inhomogeneities in bulk water that occur on femtosecond timescales. We attribute the rise in the coherent second harmonic (SH) intensity to charge density fluctuations with enhanced nanoscale polarizabilities around transient voids. Even though the transient voids also exist in carbon tetrachloride, they do not show polarizability fluctuations and the SH response of carbon tetrachloride thus corresponds to that of a model isotropic liquid. Then, we study the structure of water around large unilamellar vesicles (LUVs), which serve as a model for cell membranes. We focus on the role of water during the lipid melting phase transition. Using SHS, differential scanning calorimetry, and temperature-dependent zeta-potential measurements, we investigate the structural changes of hydration shells around the vesicle membrane. We use lipids with phosphoserine, phosphocholine, and phosphate headgroups and observe different hydration behavior upon the lipid melting transition depending on the membrane composition. Next, we characterize the interface of membranes composed of lipids containing phosphocholine and phosphate headgroups in solutions with different CaCl2 concentrations. We observe that the SH intensity is decreasing with the addition of CaCl2, which we attribute to the formation of a condensed layer of Na+ counterions on the inner leaflet of the LUVs. Additionally, we extract the vesicle surface potential and second-order surface susceptibility from SHS data for membranes with different content of lipids with phosphate headgroups, and observe composition dependent behaviour which suggests structural membrane reorganization. We continue by exploring protein-membrane interactions using water as a contrast agent. We study a pore-forming toxin, perfringolysin O (PFO), known for selective insertion into cholesterol-rich membranes. Combining SHS measurements and cryo-electron microscopy, we compare the interaction between PFO and LUV membranes with a high and low cholesterol content. By obtaining a sub-picomolar insertion constant of PFO, we show that SHS can be used to determine protein-membrane binding constants in concentration ranges not accessible by conventional methods.Finally, we investigate the interaction of protein aerolysin and its mutants with lipid membranes. We observe changes in surface charge in the cap region of membrane-bound aerolysin at different pH of the surrounding solution. By combining SHS with single-channel measurements, we obtain the membrane binding constant of wild-type aerolysin and its mutants, and we dissect their pore-forming mechanism.

EPFL2022

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ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Springer Nature2018

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