Chapter 1. Introduction
7. Lipids and membranes
7.1. Lipid metabolism and lipid rafts (LRs) formation
The membranes of eukaryoric cells contain rigid microdomains called lipid rafts (LRs), in which the GSLs and cholesterol are the key components (Lindner et al, 2009; Simons et al 2011). LRs form separate ordered structures in the matrix of the lipid bilayer, which make the membranes of eukaryotic cells not homogeneously fluid (Brown et al, 1998).
LR’s estimated size is about 5 nm in diameter, and cover as much as 35% of the cell surface (Prior et al, 2003). Distinct classes of proteins are associated with LRs and require this association as a platform to reach their proper location within the cell membrane such as glycosylphosphatidylinositol (GPI)-anchored proteins (Simons et al 2000) and intestinal disaccharidases like sucrase and isomaltase (Amiri et al 2016). The study of the LRs and the trafficking of their components have been considered as an important research area as it might serve to understand and clarify several problems associated with lipid storage diseases. Usually, LRs tend to be transient on the cell surface (Schütz et al, 2000). However, some studies have confirmed the presence of stable membrane microdomains in the endocytic pathway (Sharma et al, 2002) where they might play a role in protein and lipid sorting (Gruenberg 2001). Indeed, some studies showed that rafts can be internalized in the endocytic pathway either through clathrin-coated vesicles or other rafts pathways (Simons et al 2000). The rafts constituents (GSLs, cholesterol and GPI-anchored proteins) can be endocytosed and enter the degradative compartment but they are rapidly returned back to the cell surface via recycling endosomes (Gruenberg et al, 1995). The cell-type-specific differences should be taken into consideration in this type of studies, due to the significant variation in the endosomal organization that subsequently causes diversity in the trafficking pathway. Many trans-membrane proteins have the affinity to be ‘’raftophilic’’ and to associate with these tightly packed membrane domains (Simons et al 2011). These micro domains play an important role in post-Golgi trafficking, endocytosis, signaling in addition to other membrane functions (Simons et al, 1997). In most tissues, except the brain, the main resources
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of the cholesterol are the endogenously synthesized cholesterol and the cholesterol obtained from receptor-mediated endocytosis of plasma lipoproteins, particularly the LDL (Vance 2006). LDLs are endocytosed after binding with the cell surface LDL receptor (LDLR) and form complexes that are concentrated in the clathrin coated pits.
Upon reaching the endosomes, these complexes are disassociated due to the acidic environment in the late endosomes/lysosomes LE/L compartments. Subsequently, the acid lipase in the LE/L hydrolyzes the cholesterol esters within the LDL particles, resulting in a generation of free unesterified cholesterol (Hoeg et al, 1984). The egress of LDL- derived cholesterol out of the LE/L compartments is accomplished by the sequential action of two proteins, NPC1 and NPC2 proteins that are localized in the lysosomes (Naureckiene et al, 2000). NPC2 is a 151 amino acid small soluble protein present in the lumen of endosomes and lysosomes (Vance 2006) and could also be secreted from the cell (Carstea et al 1997; Loftus et al 1997). It has been suggested that NPC2 protein might work directly as a cholesterol transport protein (Cheruku et al, 2006). The second protein is NPC1, a polytopic transmembrane protein that is localized in late endosomes and lysosomes (Vance 2006). The orchestrated action of these two proteins allows the efflux of cholesterol from the LE/L to the other subcellular compartments including TGN, ER, mitochondria and plasma membrane (Sleat et al, 2004; Vanier 2015). Although the role of NPC1 has been well described in enabling the egress of cholesterol out of the LE/L, several studies propose additional functions of NPC1, including: cholesterol ‘flippase’ (Cruz et al, 2000) a fatty acid permease (Davies et al 2000), a ganglioside transporter (Mukherjee et al, 2004) and a cholesterol sensor that controls the export of cholesterol from the lysosomes by monitoring the level of cholesterol in these compartments (Ikonen et al, 2004). Therefore, these two proteins are considered as key players in the egress of LDL- derived cholesterol as well as other lipids out of the LE/L (Gong et al 2016).
Aim of the study
The aim of this study was to investigate the effect of various mutations on the structural features, intracellular localization and trafficking patternof the NPC1 protein in infantile, juvenile and late-onset NP-C, with the ultimate goal of defining their
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pathogenicity.These mutations are located in different regions of the protein encoding the transmembrane, luminal, cysteine-rich loop and sterol sensing domains.
The next aim was to investigate the effect of these various NPC1 mutations on the association of NPC1 mutants with lipid rafts (LRs) structure and function which might explain the variations in the disease pathomechanisms and subsequently the onset of the clinical symptoms.
This may also lead to the discovery of novel, more specific and sensitive biomarkers for diagnosing and monitoring the disease.
Since LRs are considered a potential key player in the pathogenesis of several neurodegenerative and lysosomal storage diseases, we investigated the role of LRs in the pathogenesis of Fabry disease (FD). FD is characterized by the accumulation of terminal galactosyl-containing neutral lipids due to a deficiency in the α-galactosidase (GLA) enzyme. We examined the alteration in GLA cellular trafficking and cellular membrane composition, specifically LRs, in fibroblasts from a male Fabry patient harboring the N215S mutation of the GLAgene in comparison to the wild type fibroblasts. Clinically, the patient presented an attenuated course with fatigue, arrhythmia, cardiac hypertrophy, hypothyroidism, vertigo and he required a pacemaker. Furthermore, we addressed substrate reduction therapy using Migustat in vitro, as an option to restore biochemical alterations associated with the disease, including membrane lipid composition and associated membrane proteins.
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