Proteomic identification and characterization of secreted N‐glycosylated NPC2 following cross‐linking of the high‐affinity receptor for IgE on mast cells

Allergen‐mediated cross‐linking of the high‐affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL‐2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol‐binding glycoprotein, NPC2/RE1 (Niemann–Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE‐mediated stimulus and co‐localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD‐2‐related lipid‐recognition) protein family (155 members), which includes the Toll‐like receptor co‐factors, MD‐1 and MD‐2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.     

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy,improves lysosomal function,and reduces cholesterol storage

Niemann–Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1^I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.     

Improvement in Lipid and Protein Trafficking in Niemann‐Pick C1 Cells by Correction of a Secondary Enzyme Defect

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1‐deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis‐(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.     

Telocytes: a potential defender in the spleen of Npc1 mutant mice

Niemann–Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1^?/?), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1^?/? mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c‐Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1^?/? mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1^?/? spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.     

Lipidomic Analysis Reveals Altered Fatty Acid Metabolism in the Liver of the Symptomatic Niemann–Pick,Type C1 Mouse Model

Niemann–Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result of the genetic defect, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system causing both visceral and neurological defects. These manifest clinically as hepatosplenomegaly, liver dysfunction, and neurodegeneration. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive these pathologies remains less understood. In this study, it is sought to investigate free fatty acid levels in Npc1^?/? mice with focus on the polyunsaturated ω‐3 and ω‐6 fatty acids. Since fatty acids are the main constituents of numerous lipids species, a discovery based lipidomic study of liver tissue in Npc1^?/? mice is also performed. To this end, alterations in fatty acid synthesis, including the ω‐3 and 6 fatty acids, are reported. Further, alterations in enzymes that regulate the synthesis of ω‐3 and 6 fatty acids are reported. Analysis of the liver lipidome reveals alterations in both storage and membrane lipids including ceramides, fatty acids, phosphatidylcholamines, phosphatidylglycerols, phosphatidylethanolamines, sphingomyelins, and triacylglycerols in Npc1^?/? mice at a late stage of disease.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

High‐content imaging and structure‐based predictions reveal functional differences between Niemann‐Pick C1 variants

The human Niemann‐Pick C1 (NPC1) gene encoding a 1278 amino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild‐type variant (WT‐V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe‐harbor locus on an NPC1‐null background engineered by CRISPR/Cas9 editing. We then employed high‐content imaging with automated image analysis to quantitatively assess LDL‐induced, time‐dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT‐V compromises NPC1 functionality in lysosomal cholesterol export. All‐atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C‐terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT‐V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function.     

Annexin A6 is recruited into lipid rafts of Niemann–Pick type C disease fibroblasts in a Ca-dependent manner

Niemann–Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Cholesterol as a co‐solvent and a ligand for membrane proteins

As of mid 2013 a Medline search on “cholesterol” yielded over 200,000 hits, reflecting the prominence of this lipid in numerous aspects of animal cell biology and physiology under conditions of health and disease. Aberrations in cholesterol homeostasis underlie both a number of rare genetic disorders and contribute to common sporadic and complex disorders including heart disease, stroke, type II diabetes, and Alzheimer's disease. The corresponding author of this review and his lab stumbled only recently into the sprawling area of cholesterol research when they discovered that the amyloid precursor protein (APP) binds cholesterol, a topic covered by the Hans Neurath Award lecture at the 2013 Protein Society Meeting. Here, we first provide a brief overview of cholesterol‐protein interactions and then offer our perspective on how and why binding of cholesterol to APP and its C99 domain (β‐CTF) promotes the amyloidogenic pathway, which is closely related to the etiology of Alzheimer's disease.     

Cholesterol prevents interaction of the cell‐penetrating peptide transportan with model lipid membranes

Interaction of the cell‐penetrating peptide (CPP) cysteine‐transportan (Cys‐TP) with model lipid membranes was examined by spin‐label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys‐TP on membrane structure was studied. The influence of Cys‐TP on membrane permeability was monitored by the reduction of a liposome‐trapped water‐soluble spin probe. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Mannose 6‐phosphate‐independent Lysosomal Sorting of LIMP‐2

The lysosomal integral membrane protein type 2 (LIMP‐2/SCARB2) has been described as a mannose 6‐phosphate (M6P)‐independent trafficking receptor for β‐glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP‐2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP‐2 with the cation‐independent M6P receptor (MPR) results in M6P‐dependent targeting of LIMP‐2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P‐dependent delivery of LIMP‐2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP‐2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P‐forming N‐acetylglucosamine (GlcNAc)‐1‐phosphotransferase, LIMP‐2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP‐2 levels within lysosomes purified from liver of wild‐type (wt) and GlcNAc‐1‐phosphotransferase‐defective mice. Heterologous expression of the luminal domain of LIMP‐2 in wild‐type, LIMP‐2‐deficient and GlcNAc‐1‐phosphotransferase‐defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP‐2. Finally, cathepsin Z, a known GlcNAc‐1‐phosphotransferase substrate, but not LIMP‐2, could be precipitated with M6P‐specific antibodies. These data prove M6P‐independent lysosomal sorting of LIMP‐2 and subsequently GC in vivo.      

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer’s disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1^−/− cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of lipid rafts in these processes.     

Lipid‐associated aggregate formation of superoxide dismutase‐1 is initiated by membrane‐targeting loops

Copper‐Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo‐SOD1 aggregates may be due to their ability to cause defects within cell membranes by co‐aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane‐bound copper chaperones. But how apo‐SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc‐binding loops in apo‐SOD1 dimers play a critical role in the binding of 1‐octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo‐SOD1 monomer also associates with phospholipid bilayer via its zinc‐binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane‐bound copper chaperone. The orientation also suggests how membrane‐bound monomers could act as seeds for membrane‐associated SOD1 aggregation. Proteins 2014; 82:3194–3209. © 2014 Wiley Periodicals, Inc.     

Characterization of the porcine transferrin gene (TF ) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection

Transferrin (TF)‐mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron‐depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra‐ and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed‐specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.     

Active‐Site Imprinting: Preparation of Fe–N–C Catalysts from Zinc Ion–Templated Ionothermal Nitrogen‐Doped Carbons

Atomically dispersed Fe–N–C catalysts are considered the most promising precious‐metal‐free alternative to state‐of‐the‐art Pt‐based oxygen reduction electrocatalysts for proton‐exchange membrane fuel cells. The exceptional progress in the field of research in the last ≈30 years is currently limited by the moderate active site density that can be obtained. Behind this stands the dilemma of metastability of the desired FeN₄ sites at the high temperatures that are believed to be a requirement for their formation. It is herein shown that Zn²⁺ ions can be utilized in the novel concept of active‐site imprinting based on a pyrolytic template ion reaction throughout the formation of nitrogen‐doped carbons. As obtained atomically dispersed Zn–N–Cs comprising ZnN₄ sites as well as metal‐free N₄ sites can be utilized for the coordination of Fe²⁺ and Fe³⁺ ions to form atomically dispersed Fe–N–C with Fe loadings as high as 3.12 wt%. The Fe–N–Cs are active electocatalysts for the oxygen reduction reaction in acidic media with an onset potential of E₀ = 0.85 V versus RHE in 0.1 m HClO₄. Identical location atomic resolution transmission electron microscopy imaging, as well as in situ electrochemical flow cell coupled to inductively coupled plasma mass spectrometry measurements, is employed to directly prove the concept of the active‐site imprinting approach.     

Synthesis of 3‐Methylidene‐1‐tosyl‐2,3‐dihydroquinolin‐4(1H)‐ones as Potent Cytotoxic Agents

An efficient synthetic strategy to 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones variously substituted in position 2 has been developed. The title compounds were synthesized in the reaction sequence involving reaction of diethyl methylphosphonate with methyl 2‐(tosylamino)benzoate, condensation of thus formed diethyl 2‐oxo‐2‐(2‐N‐tosylphenyl)ethylphosphonate with various aldehydes followed by successful application of the obtained 3‐(diethoxyphosphoryl)‐1,2‐dihydroquinolin‐4‐ols as Horner–Wadsworth–Emmons reagents for the olefination of formaldehyde. Also, enantioselective approach to the target compounds has been evaluated using 3‐dimenthoxyphosphoryl group as a chiral auxiliary. Single X‐ray crystal analysis of (2S)‐3‐(dimenthoxyphosphoryl)‐2‐phenyl‐1‐tosyldihydroquinolin‐4‐ol revealed the presence of strong resonance‐assisted hydrogen bond (RAHB). The obtained 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones were then tested for their cytotoxic activity against two leukemia cell lines NALM‐6 and HL‐60 and a breast cancer MCF‐7 cell line. All compounds showed very high cytotoxic activity with the IC₅₀ values mostly below 1 μm in all three cancer cell lines. The selected analogs were also tested on human umbilical vein endothelial cells (HUVEC) and on human mammary gland/breast cells (MCF‐10A) to evaluate their influence on normal cells. Since one of the most serious problems in cancer chemotherapy is the development of drug resistance, the mRNA levels and activity of ABCB1 transporter considered to be the most important factor engaged in drug resistance, were evaluated in MCF‐7 cells treated with two selected analogs. Both compounds were strong ABCB1 transporter inhibitors that could prevent efflux of anticancer drugs from cancer cells.     

Genetic differentiation of populations of Iberian rock‐lizards Iberolacerta (Iberolacerta) sensuArribas (1999)

The genetic structure and relationships of five populations of the Iberian group of Iberolacerta (sensu 6 ) were analysed by protein electrophoresis. In this study we confirmed the differentiation between the group of populations from Galicia/Cantabrian/S. Estrela versus the group of populations from de Spanish Central System, Gredos and Guadarrama that are included in the two different species by Arribas (Herpetozoa 9(1/2), 31–56, 1996; Russian J. Herpetol. 6, 1–22, 1999), Iberolacerta (I.) monticola and Iberolacerta (I.) cyreni, respectively. However, the differentiation level is not high enough to clearly prove their specific discrimination. On the other hand, we did not confirm the subspecific discrimination of the Gredos –Iberolacerta (I.) cyreni castiliana, and Guadarrama –Iberolacerta (I.) cyreni cyreni populations, proposed by 5 ). These two populations are genetically almost homogeneous. Interestingly, we found an unexpected high genetic similarity between the Galician and the Serra da Estrela populations, presently included into two different subspecies, Iberolacerta (I.) monticola cantabrica and Iberolacerta (I.) monticola monticola, respectively. Their genetic similarity is even higher than that between the populations of Galicia and the Cantabrian Mountains, which are both included in the same subspecies, cantabrica. This result suggests that the populations of Galicia and Serra da Estrela would have maintained contacts, possibly through the north of Portugal, until relatively recent times. Their separation is thus probably post‐glacial. Some evidence also points to the existence of relatively recent contacts between the population of Serra da Estrela and those of the Central System, particularly, with the neighbouring Peña de Francia population. With the cautions imposed by the reduced sample size of our analysis, the significant differentiation of the populations from Galicia and the Cantabrian Mountains allow us to suggest that this last population may not be the result of a recent expansion of the Galician population as 5 ) suggests, but, more likely, the result of a fragmentation process of a more ancient and wider north‐eastern distribution area of this group of rock‐lizards.