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Cholesterol is a structural component of cell membranes. How rapidly growing tumor cells maintain membrane cholesterol homeostasis is poorly understood. Here, we found that glioblastoma (GBM), the most lethal brain tumor, maintains normal levels of membrane cholesterol but with an abundant presence of cholesteryl esters (CEs) in its lipid droplets (LDs). Mechanistically, SREBP-1 (sterol regulatory element-binding protein 1), a master transcription factor that is activated upon cholesterol depletion, upregulates critical autophagic genes, including ATG9B, ATG4A, and LC3B, as well as lysosome cholesterol transporter NPC2. This upregulation promotes LD lipophagy, resulting in the hydrolysis of CEs and the liberation of cholesterol from the lysosomes, thus maintaining plasma membrane cholesterol homeostasis. When this pathway is blocked, GBM cells become quite sensitive to cholesterol deficiency with poor growth in vitro. Our study unravels an SREBP-1-autophagy-LD-CE hydrolysis pathway that plays an important role in maintaining membrane cholesterol homeostasis while providing a potential therapeutic avenue for GBM.

• Nine patients harbor pathological (P) or likely pathological (LP) mutations (31%) and three patients have VUS variants (10%) out of 29 DEE patients. • De novo mutations were identified in SCN2A, KCNMA1 and MECP2 genes. • Four novel variations were detected in SCN2A, SCN1A, ATP1A2 and KCNQ2 genes. • Two patients have autosomal recessive inherited mutations in NPC2 and CLN6 genes. • MD simulations revealed the destructive effects of the mutations on protein structures. In Developmental and Epileptic Encephalopathies (DEEs), identifying the precise genetic factors guides the clinicians to apply the most appropriate treatment for the patient. Due to high locus heterogeneity, WES analysis is a promising approach for the genetic diagnosis of DEE. Therefore, the aim of the present study is to evaluate the utility of WES in the diagnosis and treatment of DEE patients. The exome data of 29 DEE patients were filtrated for destructive and missense mutations in 1896 epilepsy-related genes to detect the causative variants and examine the genotype-phenotype correlations. We performed Sanger sequencing with the available DNA samples to follow the co-segregation of the variants with the disease phenotype in the families. Also, the structural effects of p.Asn1053Ser, p.Pro120Ser and p.Glu1868Gly mutations on KCNMA1, NPC2, and SCN2A proteins, respectively, were evaluated by molecular dynamics (MD) and molecular docking simulations. Out of 29, nine patients (31%) harbor pathological (P) or likely pathological (LP) mutations in SCN2A, KCNQ2, ATP1A2, KCNMA1, and MECP2 genes, and three patients have VUS variants (10%) in SCN1A and SCN2A genes. Sanger sequencing results indicated that three of the patients have de novo mutations while eight of them carry paternally and/or maternally inherited causative variants. MD and molecular docking simulations supported the destructive effects of the mutations on KCNMA1, NPC2, and SCN2A protein structures. Herein we demonstrated the effectiveness of WES for DEE with high locus heterogeneity. Identification of the genetic etiology guided the clinicians to adjust the proper treatment for the patients. [ABSTRACT FROM AUTHOR]

Niemann–Pick Disease (NPD) is a rare autosomal recessive disease belonging to lysosomal storage disorders. Three types of NPD have been described: NPD type A, B, and C. NPD type A and B are caused by mutations in the gene SMPD1 coding for sphingomyelin phosphodiesterase 1, with a consequent lack of acid sphingomyelinase activity. These diseases have been thus classified as acid sphingomyelinase deficiencies (ASMDs). NPD type C is a neurologic disorder due to mutations in the genes NPC1 or NPC2, causing a defect of cholesterol trafficking and esterification. Although all three types of NPD can manifest with pulmonary involvement, lung disease occurs more frequently in NPD type B, typically with interstitial lung disease, recurrent pulmonary infections, and respiratory failure. In this sense, bronchoscopy with broncho-alveolar lavage or biopsy together with high-resolution computed tomography are fundamental diagnostic tools. Although several efforts have been made to find an effective therapy for NPD, to date, only limited therapeutic options are available. Enzyme replacement therapy with Olipudase α is the first and only approved disease-modifying therapy for patients with ASMD. A lung transplant and hematopoietic stem cell transplantation are also described for ASMD in the literature. The only approved disease-modifying therapy in NPD type C is miglustat, a substrate-reduction treatment. The aim of this review was to delineate a state of the art on the genetic basis and lung involvement in NPD, focusing on clinical manifestations, radiologic and histopathologic characteristics of the disease, and available therapeutic options, with a gaze on future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were “Metabolic Reprogramming in Cancer” and “Glycolysis Activation in Cancer (Warburg Effect).” We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by high-resolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial-lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC.

Cholesterol is necessary for all cells to function. The intracellular cholesterol transporters Npc1 and Npc2 control sterol trafficking and their malfunction leads to Neimann–Pick Type C disease, a rare disorder affecting the nervous system and the intestine. Unlike humans that encode single Npc1 and Npc2 transporters, flies encompass two Npc1 (Npc1a-1b) and eight Npc2 (Npc2a-2h) members, and most of the Npc2 family genes remain unexplored. Here, we focus on the intestinal function of Npc2c in the adult. We find that Npc2c is necessary for intestinal stem cell (ISC) mitosis, maintenance of the ISC lineage, survival upon pathogenic infection, as well as tumor growth. Impaired mitosis of Npc2c-silenced midguts is accompanied by reduced expression of Cyclin genes, and genes encoding ISC regulators, such as Delta, unpaired1 and Socs36E. ISC-specific Npc2c silencing induces Attacin-A expression, a phenotype reminiscent of Gram-negative bacteria overabundance. Metagenomic analysis of Npc2c-depleted midguts indicates intestinal dysbiosis, whereby decreased commensal complexity is accompanied by increased gamma-proteobacteria. ISC-specific Npc2c silencing also results in increased cholesterol aggregation. Interestingly, administration of the non-steroidal ecdysone receptor agonist, RH5849, rescues mitosis of Npc2c-silenced midguts and increases expression of the ecdysone response gene Broad, underscoring the role of Npc2c and sterols in ecdysone signaling. Assessment of additional Npc2 family members indicates potential redundant roles with Npc2c in ISC control and response to ecdysone signaling. Our results highlight a previously unidentified essential role of Npc2c in ISC mitosis, as well as an important role in ecdysone signaling and microbiome composition in the Drosophila midgut. [ABSTRACT FROM AUTHOR]

Purpose of Review: A significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases. Recent Findings: More effort is dedicated toward identification of allergens' ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens' molecular and allergic properties. Summary: Four allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy. [ABSTRACT FROM AUTHOR]

Niemann–Pick Disease (NPD) is a rare autosomal recessive disease belonging to lysosomal storage disorders. Three types of NPD have been described: NPD type A, B, and C. NPD type A and B are caused by mutations in the gene SMPD1 coding for sphingomyelin phosphodiesterase 1, with a consequent lack of acid sphingomyelinase activity. These diseases have been thus classified as acid sphingomyelinase deficiencies (ASMDs). NPD type C is a neurologic disorder due to mutations in the genes NPC1 or NPC2, causing a defect of cholesterol trafficking and esterification. Although all three types of NPD can manifest with pulmonary involvement, lung disease occurs more frequently in NPD type B, typically with interstitial lung disease, recurrent pulmonary infections, and respiratory failure. In this sense, bronchoscopy with broncho-alveolar lavage or biopsy together with high-resolution computed tomography are fundamental diagnostic tools. Although several efforts have been made to find an effective therapy for NPD, to date, only limited therapeutic options are available. Enzyme replacement therapy with Olipudase α is the first and only approved disease-modifying therapy for patients with ASMD. A lung transplant and hematopoietic stem cell transplantation are also described for ASMD in the literature. The only approved disease-modifying therapy in NPD type C is miglustat, a substrate-reduction treatment. The aim of this review was to delineate a state of the art on the genetic basis and lung involvement in NPD, focusing on clinical manifestations, radiologic and histopathologic characteristics of the disease, and available therapeutic options, with a gaze on future therapeutic strategies.

In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were "Metabolic Reprogramming in Cancer" and "Glycolysis Activation in Cancer (Warburg Effect)." We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by highresolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial- lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC. [ABSTRACT FROM AUTHOR]

Cholesterol is necessary for all cells to function. The intracellular cholesterol transporters Npc1 and Npc2 control sterol trafficking and their malfunction leads to Neimann–Pick Type C disease, a rare disorder affecting the nervous system and the intestine. Unlike humans that encode single Npc1 and Npc2 transporters, flies encompass two Npc1 (Npc1a-1b) and eight Npc2 (Npc2a-2h) members, and most of the Npc2 family genes remain unexplored. Here, we focus on the intestinal function of Npc2c in the adult. We find that Npc2c is necessary for intestinal stem cell (ISC) mitosis, maintenance of the ISC lineage, survival upon pathogenic infection, as well as tumor growth. Impaired mitosis of Npc2c-silenced midguts is accompanied by reduced expression of Cyclin genes, and genes encoding ISC regulators, such as Delta, unpaired1 and Socs36E. ISC-specific Npc2c silencing induces Attacin-A expression, a phenotype reminiscent of Gram-negative bacteria overabundance. Metagenomic analysis of Npc2c-depleted midguts indicates intestinal dysbiosis, whereby decreased commensal complexity is accompanied by increased gamma-proteobacteria. ISC-specific Npc2c silencing also results in increased cholesterol aggregation. Interestingly, administration of the non-steroidal ecdysone receptor agonist, RH5849, rescues mitosis of Npc2c-silenced midguts and increases expression of the ecdysone response gene Broad, underscoring the role of Npc2c and sterols in ecdysone signaling. Assessment of additional Npc2 family members indicates potential redundant roles with Npc2c in ISC control and response to ecdysone signaling. Our results highlight a previously unidentified essential role of Npc2c in ISC mitosis, as well as an important role in ecdysone signaling and microbiome composition in the Drosophila midgut.

ABSTRACT Baculovirus is a powerful tool for biological control in agriculture and foreign gene expression and delivery in insect and mammalian cells. Baculovirus enters host cells by multiple endocytic pathways; however, the current understanding of the Bombyx mori nucleopolyhedrovirus (BmNPV) entry mechanism remains limited. Previous studies have identified NPC1 and NPC2 as important host factors for viral infection in insect cells, although their exact role in viral infection has not yet been determined. In this study, we demonstrate that the BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 in Sf9 cells confers the virus the ability to enter into the nucleus of Sf9 cells. Additionally, the second luminal domain of BmNPC1 (BmNPC1-C) binds to the viral glycoprotein gp64, and preincubation of BmNPV with purified BmNPC1-C inhibits virus infection. Furthermore, knockout of the BmNPC2 protein results in reduced efficiency of viral fusion with the endosomal membrane, and BmNPC2 protein interacts directly with both viral envelope glycoprotein gp64 and the host BmNPC1 protein. BmNPC2 was found to be incorporated into progeny viral particles. Taken together, our results suggest that NPC2 protein incorporated into viral particles may facilitate viral infection through promoting the interaction of BmNPV and NPC1 in the endosome, thus enhancing viral fusion and escape from endosomes. These results, combined with those from previous studies, support that BmNPV hijacks two important cholesterol receptor members (NPC1 and NCP2) in the cholesterol intracellular transport pathway for viral entry into host cells. IMPORTANCE Baculovirus is an important biological factor for controlling insect populations and represents a powerful biological tool for gene delivery and expression. However, the host receptor of baculovirus is still unknown. In this study, we demonstrate that BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 confers the ability of virus to enter into the host cell nucleus in nonpermissive Sf9 cells. BmNPC2 can bind to the virus and promote progeny virion infection through the NPC1-NPC2 endosome cholesterol transport pathway. We believe that our study on the BmNPV entry mechanism will further facilitate the application of baculovirus systems in eukaryotic gene delivery. Not only can the cholesterol transport pathway NPC1 protein be used by a variety of enveloped viruses, but the NPC2 protein can also be used by viruses to infect host cells. This will provide new insights into the study of enveloped virus infection mechanisms.

Steroidogenic acute regulatory (StAR)-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that facilitates cholesterol transport between cellular organelles. Cholesterol accumulation in podocytes directly contributes to the pathogenesis of albuminuria and renal injury under the condition of diabetic kidney disease (DKD). The aim of this study is to determine the role of STARD3 on the intracellular distribution of cholesterol within podocytes. In vivo and in vitro models of diabetes were performed. The protein levels of STARD3, Niemann-Pick disease type C1 (NPC1), and Niemann-Pick disease type C2 (NPC2) were respectively detected by western blot analysis, immunohistochemistry, and immunofluorescence. Filipin staining was used to evaluate the subcellular localization of cholesterol in podocytes. Mitochondrial damage was evaluated using JC-1 (CBIC2) and ROS (reactive oxygen species) assays. Upregulation of STARD3 under diabetes and hyperglycemia increases cholesterol transport from the late endosomal/lysosomal (LE/LY) to mitochondria, leading to mitochondrial cholesterol accumulation and cell injury in podocytes. Conversely, downregulating STARD3 expression attenuated mitochondrial cholesterol accumulation, and improved mitochondrial homeostasis. STARD3 may govern intracellular cholesterol transport in podocytes, subsequently leading to regulation of mitochondrial metabolism. Therefore, targeting STARD3 emerges as a potential therapeutic strategy to mitigate diabetes-induced mitochondrial cholesterol accumulation and associated injury in podocytes. Increased mitochondrial cholesterol accumulation and injury podocytes under diabetic kidney disease: STARD3 contributes to cholesterol transport from late endosomal/lysosomal (LE/LY) to the mitochondria in an NPC2-independent manner, and upregulation of STARD3 expression leads to diabetes-induced mitochondrial cholesterol accumulation and injury in podocytes. [Display omitted] [ABSTRACT FROM AUTHOR]

Sterols are an essential component of membranes in all eukaryotic cells and the precursor of multiple indispensable cellular metabolites. After endocytotic uptake, sterols are integrated into the lysosomal membrane by the Niemann‐Pick type C (NPC) system before redistribution to other membranes. The process is driven by two proteins that, together, compose the NPC system: the lysosomal sterol shuttle protein NPC2 and the membrane protein NPC1 (named NCR1 in fungi), which integrates sterols into the lysosomal membrane. The Saccharomyces cerevisiae NPC system provides a compelling model to study the molecular mechanism of sterol integration into membranes and sterol homeostasis. This review summarizes recent advances in the field, and by interpreting available structural data, we propose a unifying conceptual model for sterol loading, transfer and transport by NPC proteins. [ABSTRACT FROM AUTHOR]

Niemann Pick C2 (NPC2) is a small glycoprotein involved in cellular trafficking of cholesterol. Its dysfunction causes accumulation of cholesterol in the lysosomal organelles, a hallmark of Niemann Pick type C disease. Therefore, understanding cholesterol transport and the accumulation mechanism that may result is essential to understand the occurrence of this neurodegenerative disease. Cholestatrienol (CTL) is a fluorescent sterol widely used as an alternative to cholesterol because of its improved optical properties in spectroscopy and live-cell imaging. In this paper, the electronic circular dichroism (ECD) and the two-photon circular dichroism (TPCD) of CTL within the NPC2 binding pockets are simulated using response theory employing time-dependent density functional theory (TD-DFT). Moreover, an explicit embedding method has been used to include the effect of the protein residues and the water solvent in calculating the spectroscopic properties. In both cases, an alteration of the CD signal is observed. [ABSTRACT FROM AUTHOR]

Abstract Background The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles. Methods We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues. Results Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage. Conclusions The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability—the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses. [ABSTRACT FROM AUTHOR]

Background: Variants in Niemann-Pick Type C genes (NPC1 and NPC2) have been suggested to play a role as risk or disease modifying factors for Alzheimer's disease (AD).Objective: The aim of this study was to analyze NPC1 and NPC2 variability in demented patients with evidence of brain amyloid-β 1-42 (Aβ) deposition and to correlate genetic data with clinical phenotypes.Methods: A targeted Next Generation Sequencing panel was customized to screen NPC1, NPC2, and main genes related to neurodegenerative dementias in a cohort of 136 demented patients with cerebrospinal fluid (CSF) low Aβ levels or positive PET with Aβ tracer and 200 non-demented geriatric subjects.Results: Seven patients were carriers of NPC variants in heterozygosis. Four of them displayed pathogenic variants previously found in NPC patients and one AD patient had a novel variant. The latter was absent in 200 non-demented elderly subjects. Five of seven patients (70%) exhibited psychiatric symptoms at onset or later as compared with 43%in non-carriers (p > 0.05).Conclusion: The frequency of NPC1 and NPC2 heterozygous variants in patients with CSF evidence of Aβ deposition is higher than in the general population. [ABSTRACT FROM AUTHOR]

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability—the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a ‘Trojan horse’ therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the NPC1 or NPC2 gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of NPC2. Five-day-old npc2 mutants were morphologically indistinguishable from wildtype larvae. We found that live npc2–/– larvae exhibited stronger Nile blue staining. The npc2–/– larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the mcu (mitochondrial calcium uniporter) gene, ppp3ca (calcineurin) gene, and genes that are involved in myelination (mbp and mpz). Histological analysis of adult npc2–/– zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of il1, nfκβ, and mpeg; i.e., hallmarks of NPC disease). These findings suggest that the npc2 mutant zebrafish may be a model of NPC disease.

Glioblastoma (GBM) is one of the most common and fatal malignancies worldwide, while its prognostic biomarkers are still being explored. This study aims to identify potential genes with clinical and prognostic significance by integrating bioinformatics analysis and investigating their function in HNSCC. Based on the Single-cell RNA sequencing (scRNA-seq) results of H3K27M-glioma cells, computational bioinformatics methods were employed for selecting prognostic biomarker for GBM. The protein NPC2 (NPC Intracellular Cholesterol Transporter 2), which has been shown to be related to lipoprotein metabolism and innate immune system, was identified to be upregulated in GBM. NPC2 showed a relatively higher expression in GBM samples, and a negative correlation with tumor purity and tumor infiltrating immune cells. Additionally, NPC2 was knocked down in U87-MG and U251 cells line, and cell proliferation and migration capability were evaluated with CCK-8, scratch and transwell assay, respectively. Cytological experiments has shown that NPC2 overexpression inhibited GBM cells proliferation and migration, indicating its important role in GBM progression. This is the first investigation into the prognostic value of NPC2 interact with GBM. The potential molecular factor NPC2 have been identified as a prognostic biomarker for GBM.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the NPC1 or NPC2 gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of NPC2. Five-day-old npc2 mutants were morphologically indistinguishable from wildtype larvae. We found that live npc2–/– larvae exhibited stronger Nile blue staining. The npc2–/– larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the mcu (mitochondrial calcium uniporter) gene, ppp3ca (calcineurin) gene, and genes that are involved in myelination (mbp and mpz). Histological analysis of adult npc2–/– zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of il1 , nf κβ, and mpeg ; i.e., hallmarks of NPC disease). These findings suggest that the npc2 mutant zebrafish may be a model of NPC disease. [ABSTRACT FROM AUTHOR]

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stageswasmilder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed inMZnpc2m/m zebrafish. [ABSTRACT FROM AUTHOR]

The lysosomal membrane protein Niemann‐Pick type C1 (NPC1) and Niemann‐Pick type C2 (NPC2) are main players of cholesterol control in the lysosome and it is known that the mutation on these proteins leads to the cholesterol trafficking‐related neurodegenerative disease, which is called the NPC disease. The mutation R518W or R518Q on the NPC1 is one of the type of disease‐related mutation that causes cholesterol transports to be cut in half, which results in the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment of the cell. Even though there has been significant progress with understanding the cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of the full‐length NPC1 in 2016, many details such as the interaction of the full‐length NPC1 with the NPC2, the molecular motions responsible for the cholesterol transport during and after this interaction, and the structure and the function relations of many mutations are still not well understood. In this study, we report the extensive molecular dynamics simulations in order to gain insight into the structure and the dynamics of NPC1 lumenal domain for the cholesterol transport and the disease behind the mutation (R518W). It was found that the mutation induces a structural shift of the N‐terminal domain, toward the loop region in the middle lumenal domain, which is believed to play a central role in the interaction with NPC2 protein, so the interaction with the NPC2 protein might be less favorable compared to the wild NPC1. Also, the simulation indicates the possible re‐orientation of the N‐terminal domain with both the wild and the R518W‐mutated NPC1 after receiving the cholesterol from the NPC2 that align to form an internal tunnel, which is a possible pose for further action in cholesterol trafficking. We believe the current study can provide a better understanding of the cholesterol transport by NPC1 especially the role of NTD of NPC1 in combination with NPC2 interactions. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is one of the most common and fatal malignancies worldwide, while its prognostic biomarkers are still being explored. This study aims to identify potential genes with clinical and prognostic significance by integrating bioinformatics analysis and investigating their function in HNSCC. Based on the Single-cell RNA sequencing (scRNA-seq) results of H3K27M-glioma cells, computational bioinformatics methods were employed for selecting prognostic biomarker for GBM. The protein NPC2 (NPC Intracellular Cholesterol Transporter 2), which has been shown to be related to lipoprotein metabolism and innate immune system, was identified to be upregulated in GBM. NPC2 showed a relatively higher expression in GBM samples, and a negative correlation with tumor purity and tumor infiltrating immune cells. Additionally, NPC2 was knocked down in U87-MG and U251 cells line, and cell proliferation and migration capability were evaluated with CCK-8, scratch and transwell assay, respectively. Cytological experiments has shown that NPC2 overexpression inhibited GBM cells proliferation and migration, indicating its important role in GBM progression. This is the first investigation into the prognostic value of NPC2 interact with GBM. The potential molecular factor NPC2 have been identified as a prognostic biomarker for GBM. [ABSTRACT FROM AUTHOR]

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease., Competing Interests: YZ, MZ, HW, XW, HZ, JF, JW, LL, HX, CQ, XL, YZ, WH, YW, YW, YS, JF, GC No competing interests declared, (© 2023, Zhang, Zhang, Wu et al.)

Defective mitochondria are pathophysiological features of a number of neurodegenerative diseases. Here, we investigated mitochondrial dysfunction in the context of the rare lysosomal storage diseases Niemann–Pick disease type C1 and type C2 (NP-C1 and NP-C2). Mutations in either the NPC1 or NPC2 gene lead to cholesterol accumulation in late endosomes and lysosomes, resulting in impaired cholesterol homeostasis. The extent to which this may lead to mitochondrial dysfunction has been poorly studied so far. Therefore, we investigated the morphology, function, and transport of mitochondria, as well as their degradation via mitophagy, in a disease-associated human neural cell model of NP-C. By performing live cell imaging, we observed markedly reduced mitochondrial transport, although morphology and function were not appreciably altered. However, we observed a defective mitophagy induction shown by a reduced capability to elevate parkin expression and engulf mitochondria in autophagosomes after treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This was accompanied by defects in autophagy induction, exhibited by a hampered p62 expression and progression, shown by increased LC3BII levels and a defective fusion of autophagosomes and lysosomes. The latter might have been additionally influenced by the observed reduced lysosomal transport. Hence, we hypothesized that a reduced recycling of mitochondria contributes to the pathophysiology of NP-C.

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Variation between and within boar ejaculates in terms of their ability to withstand freeze-thawing is a limitation for sperm cryopreservation. Consequently, searching for freezability markers not only in sperm but also in seminal plasma (SP) is imperative. The present study aimed to evaluate the relationship between cholesterol content, relative levels of NPC2 and AQN-1 at two different holding times (0 h: HT0 and 24 h: HT24) at 17 °C, and boar sperm freezability. Forty-five ejaculates were cryopreserved and subsequently classified as of good (GFE) or poor (PFE) freezability according to their post-thaw sperm viability and total motility. Prior to cryopreservation, relative abundances of two SP proteins (NPC2 and AQN-1) and cholesterol content in sperm and SP were determined through immunoblotting and colorimetric methods, respectively. These determinations were made after ejaculation (HT0) and after 24 h of storage at 17 °C (HT24). Two bands for NPC2 protein (16 kDa and 19 kDa) were identified. Relative amounts of the 16 kDa-band were significantly (P < 0.05) higher in poor (PFE) than in good (GFE) freezability ejaculates both at HT0 and HT24, whereas those of the 19 kDa-band were significantly (P < 0.05) higher in PFE than in GFE at HT24 only. In the case of AQN-1, no significant differences between GFE and PFE were observed. In addition, no variations in the cholesterol content of sperm and SP were observed either between HT0 and HT24 or between GFE and PFE. We can conclude that the content of two NPC2 isoforms in SP, but not of that of spermadhesin AQN-1, may be involved in the sperm resilience to withstand freeze-thawing procedures and may predict ejaculate freezability. While a possible mechanism through which NPC2 during HT could affect boar sperm cryotolerance is suggested to be related to its ability to bind the plasma membrane cholesterol, further research is warranted. • Two NPC2 isoforms (16 kDa and 19 kDa) were identified in boar seminal plasma. • Relative contents of NPC2 isoforms in fresh semen are related to sperm cryotolerance. • Spermadhesin AQN-1 content in SP is not related with sperm cryotolerance. • Further research on the NPC2 mechanisms improving sperm cryotolerance is warranted. [ABSTRACT FROM AUTHOR]

Background: The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles. Methods: We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues. Results: Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage. Conclusions: The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts. [ABSTRACT FROM AUTHOR]

External validation in different cohorts is a key step in the translational development of new biomarkers. We previously described three host mRNA whose expression in peripheral blood is significantly higher (NPC2) or lower (DOCK9 and EPHA4) in individuals with TB compared to latent TB infection (LTBI) and controls. We have now conducted an independent validation of these genes by re-analyzing publicly available transcriptomic datasets from Brazil, China, Haiti, India, South Africa, and the United Kingdom. Comparisons between TB and control/LTBI showed significant differential expression of all three genes (NPC2high p < 0.01, DOCK9low p < 0.01, and EPHA4low p < 0.05). NPC2high had the highest mean area under the ROC curve (AUROC) for the differentiation of TB vs. controls (0.95) and LTBI (0.94). In addition, NPC2 accurately distinguished TB from the clinically similar conditions pneumonia (AUROC, 0.88), non-active sarcoidosis (0.87), and lung cancer (0.86), but not from active sarcoidosis (0.66). Interestingly, individuals progressing from LTBI to TB showed a constant increase in NPC2 expression with time when compared to non-progressors (p < 0.05), with a significant change closer to manifestation of active disease (≤3 months, p = 0.003). Moreover, NPC2 expression normalized with completion of anti-TB treatment. Taken together, these results validate NPC2 mRNA as a diagnostic host biomarker for active TB independent of host genetic background. Moreover, they reveal its potential to predict progression from latent to active infection and to indicate a response to anti-TB treatment.

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

Cancer's high incidence and death rate jeopardize human health and life, and it has become a global public health issue. Some members of NPCs have been studied in a few cancers, but comprehensive and prognostic analysis is lacking in most cancers. In this study, we used the Cancer Genome Atlas (TCGA) data genomics and transcriptome technology to examine the differential expression and prognosis of NPCs in 33 cancer samples, as well as to investigate NPCs mutations and their effect on patient prognosis and to evaluate the methylation level of NPCs in cancer. The linked mechanisms and medication resistance were subsequently investigated in order to investigate prospective tumor therapy approaches. The relationships between NPCs and immune infiltration, immune cells, immunological regulatory substances, and immune pathways were also investigated. Finally, the LUAD and KICH prognostic prediction models were built using univariate and multivariate COX regression analysis. Additionally, the mRNA and protein levels of NPCs were also identified.

Introduction: The growing number of genetic variants of unknown significance (VUS) and availability of several in silico prediction tools make the evaluation of potentially deleterious gene variants challenging.Materials and Methods: We evaluated several programs and software to determine the one that can predict the impact of genetic variants found in lysosomal storage disorders (LSDs) caused by defects in cholesterol trafficking best. We evaluated the sensitivity, specificity, accuracy, precision, and Matthew's correlation coefficient of the most common software.Results: Our findings showed that for exonic variants, only MutPred1 reached 100% accuracy and generated the best predictions (sensitivity and accuracy = 1.00), whereas intronic variants, SROOGLE or Human Splicing Finder (HSF) generated the best predictions (sensitivity = 1.00, and accuracy = 1.00).Discussion: Next-generation sequencing substantially increased the number of detected genetic variants, most of which were considered to be VUS, creating a need for accurate pathogenicity prediction. The focus of the present study is the importance of accurately predicting LSDs, with majority of previously unreported specific mutations.Conclusion: We found that the best prediction tool for the NPC1, NPC2, and LIPA variants was MutPred1 for exonic regions and HSF and SROOGLE for intronic regions and splice sites. [ABSTRACT FROM AUTHOR]

Niemann-Pick type C2 disease (NP-C2) is a fatal neurovisceral disorder caused by defects in the lysosomal cholesterol transporter protein NPC2. Consequently, cholesterol and other lipids accumulate within the lysosomes, causing a heterogeneous spectrum of clinical manifestations. Murine models are essential for increasing the understanding of the complex pathology of NP-C2. This study, therefore, aims to describe the neurovisceral pathology in the NPC2-deficient mouse model to evaluate its correlation to human NP-C2. Npc2 −/− mice holding the LST105 mutation were used in the present study (Npc2 Gt(LST105)BygNya ). Body and organ weight and histopathological evaluations were carried out in six and 12-week-old Npc2 −/− mice, with a special emphasis on neuropathology. The Purkinje cell (PC) marker calbindin, the astrocytic marker GFAP, and the microglia marker IBA1 were included to assess PC degeneration and neuroinflammation, respectively. In addition, the pathology of the liver, lungs, and spleen was assessed using hematoxylin and eosin staining. Six weeks old pre-symptomatic Npc2 −/− mice showed splenomegaly and obvious neuropathological changes, especially in the cerebellum, where initial PC loss and neuroinflammation were evident. The Npc2 −/− mice developed neurological symptoms at eight weeks of age, severely progressing until the end-stage of the disease at 12 weeks. At the end-stage of the disease, Npc2 −/− mice were characterized by growth retardation, tremor, cerebellar ataxia, splenomegaly, foam cell accumulation in the lungs, liver, and spleen, brain atrophy, pronounced PC degeneration, and severe neuroinflammation. The Npc2 Gt(LST105)BygNya mouse model resembles the pathology seen in NP-C2 patients and denotes a valuable model for increasing the understanding of the complex disease manifestation and is relevant for testing the efficacies of new treatment strategies. • The Npc2 -/- mice show neurovisceral pathology like Niemann Picks disease type C in humans and resemble the juvenile form • Neuropathological changes are evident in the cerebellum weeks before the onset of neurological symptoms • Purkinje cells show pronounced axonal swellings at the end stage of the disease • Npc2 -/- mice are characterized by tremors, ataxia, Purkinje cell death, widespread neuroinflammation, and cholesterol storage [ABSTRACT FROM AUTHOR]

Abstract The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre‐cancerous lesions remains elusive. We have used the V600EBRAF‐driven mouse lung model that develop premalignant lesions to understand stroma–tumour interactions during pre‐cancerous development. In this model, we have found that immature macrophage‐lineage cells (IMCs) producing PDGFA, TGFβ and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)‐dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial–mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early‐stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol‐binding protein, Niemann‐Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer.

Niemann-Pick type C2 (NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of low-density lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2 (NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor (LTβR) activation or Baff receptor (BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system. [ABSTRACT FROM AUTHOR]

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association. [ABSTRACT FROM AUTHOR]

Major domestic mite allergens are present in feces. We present a detailed 2D–E-MS/MS proteomic analysis of the Dermatophagoides pteronyssinus feces. Precise cultivation yielded a pure fecal extract. We detected differences in fecal allergens/digestive enzymes between D. pteronyssinus and D. farinae using 2D–E fingerprinting, including unique information on species-specific protease isoforms. Proteomic analysis was performed by 2D–E coupled with MALDI-TOF/TOF identification. The species-specific differences in the fecal extracts of the mites were attributed to trypsin-like proteases known as group 3 allergens. In D. farinae , Der f 3 exhibited high abundance with a pI similar (acidic) to that of the cysteine protease Der f 1 and the chymotrypsin protease Der f 6, whereas in D. pteronyssinus , Der p 3 was rarely detected and exhibited low abundance only at basic pI. Moreover, Der p 9 was detected at a pI of ~ 10, in contrast to Der p 1 and Der p 6, suggesting different compartmentalization in the body. Overall, in D. pteronyssinus feces, allergens of groups 1, 2, 6, and 15 were quantitatively similar to those of D. farinae with the exception of the group 3 and 9 allergens. This work provides novel insights into mite-defecated proteins/digestive enzymes, which are important allergens. Significance Millions of people are affected by allergy and asthma, and their number is growing. In homes, the major triggers of allergy and asthma are the house dust mites Dermatophagoides farinae and D. pteronyssinus , and a clear understanding of the development of diseases caused by these mites is needed. The major sources of mite allergens are their feces, which are deposited in the environment and are easily inhaled as part of aeroplankton. However, descriptions of and comparisons between the major fecal allergens of these two mites are lacking. This study shows that similar group 1 (cysteine protease), 2 (NPC2 family), 6 (chymotrypsin) and 15 (chitinase-like) allergens are present in the feces of these two mite species, as determined by 2D–E mapping, whereas group 3 (trypsin) and 9 (collagenolytic protease) allergens in the feces of the two species are different. The results provide unique MS/MS mapped fingerprints of mite species-specific isoforms in feces. The presence of ubiquitin in mite feces suggests that these proteins participate in the post-translational modification of fecal proteins. The findings are essential for understanding differences between D. farinae and D. pteronyssinus with respect to immunoreactivity, protease activation mechanisms, association with microbes, and food utilization. [ABSTRACT FROM AUTHOR]

Mammalian cells acquire most exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co-ordinated actions of Niemann-Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post-endosomal cholesterol trafficking. Numerous sterol-transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome-peroxisome membrane contact (LPMC) established by a non-STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5-bisphosphate also serves as a novel and important path for LDL-cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Objective: To describe the clinical features and course of liver involvement in a cohort of patients with Niemann-Pick type C disease (NP-C), a severe lysosomal storage disorder.Study Design: Patients with genetically confirmed NP-C (NPC1, n = 31; NPC2, n = 3) and liver involvement before age 6 months were retrospectively included. Clinical, laboratory test, and imaging data were collected until the last follow-up or death; available liver biopsy specimens were studied using anti-CD68 immunostaining.Results: At initial evaluation (median age, 17 days of life), all patients had hepatomegaly, 33 had splenomegaly, and 30 had neonatal cholestasis. Portal hypertension and liver failure developed in 9 and 4 patients, respectively. Liver biopsy studies, performed in 16 patients, revealed significant fibrosis in all 16 and CD68+ storage cells in 15. Serum alpha-fetoprotein concentration measured in 21 patients was elevated in 17. Plasma oxysterol concentrations were increased in the 16 patients tested. Four patients died within 6 months of life, including 3 from liver involvement. In patients who survived beyond age 6 months (median follow-up, 6.1 years), cholestasis regressed in all, and portal hypertension regressed in all but 1; 25 patients developed neurologic involvement, which was fatal in 16 patients.Conclusions: Liver involvement in NP-C consisted of transient neonatal cholestasis with hepatosplenomegaly, was associated with liver fibrosis, and was responsible for death in 9% of patients. The combination of liver anti-CD68 immunostaining, serum alpha-fetoprotein measurement, and studies of plasma biomarkers should facilitate early identification of NP-C. [ABSTRACT FROM AUTHOR]

Niemann-Pick type C (NP-C) disease is a neurovisceral disorder caused by mutations in the NPC1 and NPC2 genes. It is characterized by lysosomal storage of a broad range of lipids as a result of abnormal intracellular lipid trafficking. Typically patients develop neurodegeneration; however, the speed of disease progression is variable. The exact functions of NPC1 and NPC2 proteins have not been determined and therefore the molecular pathophysiology of NP-C is still not clearly understood. Due to the disease's rarity and clinical heterogeneity, delays from symptom onset to diagnosis and treatment initiation are common. Current therapeutic approaches focus on multidisciplinary symptom control and deceleration (rather than reversal) of disease progression. Thus identification of cases at early stages of disease is particularly important. Recent advances in genetic and biochemical testing have resulted in the generation of relatively non-invasive, quick and cost-effective laboratory assays that are highly sensitive and specific and have the capacity to enhance the clinicians' ability to reach a diagnosis earlier. Miglustat is a compound recently licensed in many countries for the treatment of NP-C that has been shown to decelerate neurological regression, whereas many other promising drugs are currently being trialled in preclinical models or human studies. This review summarizes key clinical, genetic and biochemical features of NP-C, suggests a simple diagnostic investigation strategy and gives an overview of available therapeutic options as well as potential novel treatments currently under development. [ABSTRACT FROM AUTHOR]

Niemann-pick type C1 ( NPC1) is an endo/lysosomal membrane protein involved in intracellular cholesterol trafficking, and its luminal domain C is an essential endosomal receptor for Ebola and Marburg viruses. We have determined the crystal structure of glycosylated NPC1 luminal domain C and find all seven possible sites are glycosylated. Mapping the disease mutations onto the glycosylated structure reveals a potential binding face for NPC2. Knowledge-based docking of NPC1 onto Ebola viral glycoprotein and sequence analysis of filovirus susceptible and refractory species reveals four critical residues, H418, Q421, F502 and F504, some or all of which are likely responsible for the species-specific susceptibility to the virus infection. [ABSTRACT FROM AUTHOR]

Background & Aims Niemann-Pick C2 (NPC2) is a lysosomal protein involved in the egress of low-density lipoprotein-derived cholesterol from lysosomes to other intracellular compartments. NPC2 has been detected in several tissues and is also secreted from the liver into bile. We have previously shown that NPC2-deficient mice fed a lithogenic diet showed reduced biliary cholesterol secretion as well as cholesterol crystal and gallstone formation. This study aimed to investigate the consequences of NPC2 hepatic overexpression on liver cholesterol metabolism, biliary lipid secretion, gallstone formation and the effect of NPC2 on cholesterol crystallization in model bile. Methods We generated NPC2 transgenic mice ( Npc2.Tg ) and fed them either chow or lithogenic diets. We studied liver cholesterol metabolism, biliary lipid secretion, bile acid composition and gallstone formation. We performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. Results No differences were observed in biliary cholesterol content or secretion between wild-type and Npc2.Tg mice fed the chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but did not show differences in the bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. Finally, recombinant NPC2 decreased nucleation time in model bile. Conclusions These results suggest that NPC2 promotes cholesterol gallstone formation by decreasing the cholesterol nucleation time, indicating a pro-nucleating function of NPC2 in bile. [ABSTRACT FROM AUTHOR]

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.

Introduction: NPC2 is well known as a player for cholesterol transport. However, the biological role of NPC2 in cancer development and therapy is far from clear., Methods: Here, we explore the potential role of NPC2 in prognosis and immunotherapy across multiple cancer types by integrating RNA-seq data from TCGA and GTEx, protein data from CPTAC, and multiple web analysis databases., Results: Expression depiction between tumour and normal tissues indicated that NPC2 is overexpressed in the majority of the most common cancer types, including glioblastoma and pancreatic cancer, two cancers mostly difficult to diagnose and treat., Conclusion: Cancer stemness in glioblastoma is negatively associated with NPC2 level. NPC2 expression is positively correlated with immune cell infiltration and the expression of several immune checkpoints. IDH1 mutation in GBM is negatively correlated with NPC2 level, while a positive correlation has been found between TP53 mutation and NPC2 expression in pancreatic cancer. NPC2 is also correlated with levels of serum biomarkers used for diagnosis of pancreatic cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)