2.A.6.6.1
Niemann-Pick C1 (SLC65A1) and C2 disease proteins, NPC1 and NPC2, together may form a lipid/cholesterol exporter from lysosomes to other cellular sites including the plasma membrane (Sleat et al., 2004; Kennedy et al. 2014). NPC1 or NPC2 deficiency causes lysosomal retention of cholesterol, sphingolipids, phospholipids, and glycolipids as well as neuronal dysfunction and neurodegeneration (Infante et al. 2008a). Cholesterol binds to the sterol-sensing domain (Ohgami et al. 2004). Increased mitochondrial cholesterol, observed in NPC1 or NPC2 deficiency, causes oxidative stress and increased rates of glycolysis and lactate release (Kennedy et al. 2014).  NPC1 binds cholesterol, 25-hydroxycholesterol and various oxysterols (Infante et al. 2008b; Liu et al., 2009 ). Soluble NPC2 binds cholesterol, and then passes it to the N-terminal domain of membranous NPC1 (Abi-Mosleh et al., 2009). Cholesterol trafficking in Niemann-Pick C-deficient cells was reviewed by Peake and Vance (2010). NPC1 is a late-endosomal membrane protein involved in trafficking of LDL- derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection.  It is the Ebola virus receptor. It contains 13 TMSs, five of which are thought to represent a "sterol-sensing domain", also present in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling. A crystal structure of a large fragment of human NPC1 at 3.6 Å resolution revealed internal twofold pseudosymmetry along TMSs 2-13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen (Li et al. 2016). NPC1's sterol sensing domain forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; this cavity is large enough to accommodate one cholesterol molecule. A model was proposed for  cholesterol sensing and transport (Li et al. 2016).  Lysosomal cholesterol activates TORC1 via an SLC38A9-Niemann-Pick C1 signaling complex (Castellano et al. 2017).  Gong et al. 2016 presented a 4.4 Å structure of the full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains three distinct lumenal domains A (also designated NTD), C, and I. TMSs 2-13 exhibit a typical RND fold, among which TMSs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through domain C (Gong et al. 2016). The effects of disease-causing mutations on quality control pathways involving the lysosome and endoplasmic reticulum, and how it functions to clear the most common mutant protein found in Niemann-Pick type C patients have been reviewed (Schultz et al. 2016). In the same review, knowledge concerning the mechanisms that degrade misfolded transmembrane proteins in the endoplasmic reticulum is presented. Cholesterol esters are components of low density lipoprotein (LDL), which is brought into the cells of various tissues by targeted endocytosis. Within the endosomes, cholesterol esters are hydrolyzed, releasing free cholesterol, which is finally exported out of the endosome by NPC1 with assistance from a soluble protein NPC2 (Nikaido 2018). The transmembrane helices in the N-terminal half (the SSD, sterol-sensing domain) of NPC1 are homologous to the sterol-binding domains of HMG-CoA reductase, as well as the regulator of cholesterol-regulated transcription activation, SCAP. The domain that binds cholesterol with the highest affinity, within NPC1, however, is the NTD. In the NPC2-NPC1 complex, the substrate is captured at a location far away from the membrane by NPC2, and then is brought to a location close to the membrane surface (NTD of NPC1), and is finally moved to the intramembranous region of NPC1. Degradation occurs via two pathways, the proteasome following MARCH6-dependent ERAD, and an autophagic pathway called the selective ER autophagy (ER-phagy) (Schultz et al. 2018). NPC1 exports LDL-derived cholesterol from lysosomes by carrying it through the 80 Å glycocalyx and the 40 Å lipid bilayer. Transport begins when cholesterol binds to the N-terminal domain (NTD) of NPC1, which projects to the surface of the glycocalyx. Trinh et al. 2018 reconstituted cholesterol transport by expressing the NTD as a fragment separate from the remaining portion of NPC1. When co-expressed, the two NPC1 fragments reconstitute cholesterol transport and showed that cholesterol can be transferred from the NTD of one full-length NPC1 to another NPC1 molecule that lacks the NTD. The locations of buried amino acids and docking studies have identified putative lipid binding domains that are in close proximity to amino acids that, when mutated, are connected to NPC1 loss-of-function (Elghobashi-Meinhardt 2019). Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding, and lysosomal cholesterol egress requires both NPC1 and NPC2. Qian et al. 2020 presented systematic structural characterizations that revealed the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A cholesterol molecule was found at the membrane boundary of the tunnel, and TMS2 moves toward formation of a surface pocket on the SSD. The structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD) (Qian et al. 2020). Genetic diversity in Niemann-Pick C1 can be managed through modulation of the Hsp70 chaperone system (Wang et al. 2020). The NPC1 protein is evolutionarily conserved with homologues reported in yeast to humans; NPC2 is present in C. elegans to humans. While neurons in mammalian models of NPC1 and NPC2 diseases exhibit many changes that are similar to those in humans (e.g., endosomal/lysosomal storage, Golgi fragmentation, neuroaxonal dystrophy, neurodegeneration), a reduced degree of ectopic dendritogenesis and an absence of neurofibrillary tangles (NFTs) in these species suggest important differences in the way lower mammalian neurons respond to NPC1/NPC2 loss of function (Walkley and Suzuki 2004). Cholesterol transport studies using wild-type NPC1 and the P691S mutant suggest changes in dynamical behavior as determined using molecular dynamics simulations (Elghobashi-Meinhardt 2020). NPC1 mutations cause variable disease phenotypes (Musalkova et al. 2020). Filoviruses, including marburgviruses and ebolaviruses, have a single transmembrane glycoprotein (GP) that facilitates their entry into cells. During entry, GP needs to be cleaved by host proteases to expose the receptor-binding site that binds to the endosomal receptor Niemann-Pick C1 (NPC1) protein. Crystal structural analyses of the cleaved GP (GPcl) of Ebola virus (EBOV) in complex with human NPC1 has shown that NPC1 has two protruding loops (loops 1 and 2), which engage a hydrophobic pocket on the head of EBOV GPcl (Igarashi et al. 2021). Cholesterol docking studies, focusing on binding recognition, showed differences in the binding positions of mutant variants versus the wild-type protein (Martínez-Archundia et al. 2020). Commonalities between Niemann-Pick C1 disease and other lysosomal storage disorders have been reviewed (Yañez et al. 2020). Cholesterol binding to the sterol-sensing region of the Niemann Pick C1 protein confines dynamics of its N-terminal domain (Dubey et al. 2020). Variants in the Niemann-Pick type C gene, NPC1, are probably not associated with Parkinson's disease (Ouled Amar Bencheikh et al. 2020). NPC1 exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMSs 3-7 of NPC1 comprise the Sterol-Sensing Domain (SSD). The anti-fungal drug itraconazole abolishes NPC1 activity in cells. Long et al. 2020 reported a cryo-EM structure of human NPC1 bound to itraconazole, which reveals how this binding site in the center of NPC1 blocks a putative lumenal tunnel linked to the SSD. Blocking this tunnel abolishes NPC1-mediated cholesterol egress. The palmitate anchor of Hedgehog occupies a similar site in the homologous tunnel of Patched, suggesting a conserved mechanism for sterol transport in this family of proteins and establishing a central function of their SSDs. Npc1, acting in neurons and glia, is essential for the formation and maintenance of CNS myelin (Yu and Lieberman 2013). In contrast to the benign Q92S mutation, Q92R reduces electrostatic potential around S-opening, and thus likely affects the NPC1 (NTD)-NPC2 interaction and/or cholesterol transfer from NPC2 to NPC1 (Petukh and Zhulin 2018). In the somatosensory neocortex, NPC1 plays a role in synaptic function (Avchalumov et al. 2012). Lysosomes receive extracellular and intracellular cholesterol and redistribute it throughout the cell. Cholesterol egress from lysosomes is critical for cholesterol homeostasis, and its failure underlies the pathogenesis of genetic disorders such as Niemann-Pick C disease. Anderson et al. 2022 reported that the BORC-ARL8-HOPS ensemble is required for egress of free cholesterol from lysosomes and for storage of esterified cholesterol in lipid droplets. Depletion of BORC, ARL8 or HOPS does not alter the localization of the lysosomal transmembrane cholesterol transporter NPC1 to degradative compartments, but decreases the association of the luminal transporter NPC2 and increases NPC2 secretion. BORC-ARL8-HOPS depletion also increases lysosomal degradation of CI-MPR, which normally sorts NPC2 to the endosomal-lysosomal system and then is recycled to the trans-Golgi network (TGN). These defects likely result from impaired HOPS-dependent fusion of endosomal-lysosomal organelles and an uncharacterized function of HOPS in CI-MPR recycling. Thus, the BORC-ARL8-HOPS ensemble is required for cholesterol egress from lysosomes by enabling CI-MPR-dependent trafficking of NPC2 to the endosomal-lysosomal system (Anderson et al. 2022). Members of this familly have the NPC1 fold (Ferrada and Superti-Furga 2022).