Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.     

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.     

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.     

The nucleoporin Nup358/RanBP2 promotes nuclear import in a cargo- and transport receptor-specific manner

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/β-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Niemann–Pick C1-Like 1 and cholesterol uptake

Niemann–Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

Epitope mapping of gp350/220 conserved domain of epstein barr virus to develop nasopharyngeal carcinoma (npc) vaccine

Nasopharyngeal carcinoma (NPC) is a malignant tumor in the nasopharyngeal epithelial cells that caused by many factors, one of which is the viral infection of EBV (Epstein Barr Virus). The standard treatments to cure NPC still have not been encouraging. The prevention through vaccination is an effective way to stop the disease. However, EBV vaccine being able to cover all variants of virus is still not available yet. Therefore, we identified the conserved region of glycoprotein 350/220 of EBV which has immunogenic and antigenic properties. The glycoprotein 350/220 is viral surface protein responsible to bind CR2 receptor, mediated EBV to enter the host cell. The conserved domain is crucial for EBV in infecting host cells. Further, by blocking CR2 binding domain of gp350/220 using antibody will inhibit EBV's spreading, and provoke an immune system to eliminate the virus in a patient. Glycoprotein 350/220 from all variants of Epstein-Barr virus was retrieved from NCBI. The conserved domain of gp350/220 was identified by aligning the protein sequences and structures. The polymorphic structure was used as a template for docking analysis to identify the resemblance of amino acid from polymorphic variants of gp350/220 that binds CR2. The epitope mapping of gp350/220 was done by Discotope BepiPred method. The result revealed that the conserved region of gp350/220 was predicted to have an epitope, QNPVYLIPETVPYIKWDNC residue, and it does not have any similarities to the human's cell surface protein. Therefore, it can be used as a reference to develop vaccine to prevent NPC.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.