Niemann-Pick C1 Protein Research Articles

Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates, and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro in mouse TNBC xenografts and in patient-derived estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single-agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol-trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Significance: Two FDA-approved compounds, AKT inhibitors and pitavastatin, synergize to induce cell death in triple-negative breast cancer, motivating evaluation of the efficacy of this combination in clinical trials.

In silico comparative analysis of cestode and human NPC1 provides insights for ezetimibe repurposing to visceral cestodiases treatment

Visceral cestodiases, like cysticercoses and echinococcoses, are caused by cystic larvae from parasites of the Cestoda class and are endemic or hyperendemic in many areas of the world. Current therapeutic approaches for these diseases are complex and present limitations and risks. Therefore, new safer and more effective treatments are urgently needed. The Niemann-Pick C1 (NPC1) protein is a cholesterol transporter that, based on genomic data, would be the solely responsible for cholesterol uptake in cestodes. Considering that human NPC1L1 is a known target of ezetimibe, used in the treatment of hypercholesterolemia, it has the potential for repurposing for the treatment of visceral cestodiases. Here, phylogenetic, selective pressure and structural in silico analyses were carried out to assess NPC1 evolutive and structural conservation, especially between cestode and human orthologs. Two NPC1 orthologs were identified in cestode species (NPC1A and NPC1B), which likely underwent functional divergence, leading to the loss of cholesterol transport capacity in NPC1A. Comparative interaction analyses performed by molecular docking of ezetimibe with human NPC1L1 and cestode NPC1B pointed out to similarities that consolidate the idea of cestode NPC1B as a target for the repurposing of ezetimibe as a drug for the treatment of visceral cestodiases.

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

Erratum for the Report “Transmembrane molecular pump activity of Niemann-Pick C1 protein,” by J. P. Davies et al .

Erratum for the Report “Transmembrane molecular pump activity of Niemann-Pick C1 protein,” by J. P. Davies <i>et al</i> .

Upregulation of NPC1 and its association with poor prognosis in gastric cancer.

The Niemann-Pick disease type C1 (NPC1) protein plays a pivotal role in lipid transport, particularly free cholesterol, within lysosomal/late endosomal membranes. Previous studies have highlighted NPC1 as a promising target for cholesterol trafficking and cancer therapy. Nevertheless, the expression of NPC1 in gastric cancer (GC) and its clinical implications remain unexplored. This study aims to investigate NPC1 expression in GC and its correlation with patient prognosis. NPC1 expression levels in GC and normal tissues were assessed using the GEPIA database, and survival analysis was conducted via Kaplan‒Meier Plotter. Evaluation of potential biological effects of NPC1 in GC by protein-protein interaction network and GO, KEGG bioenrichment analysis. Immunohistochemistry was performed on surgical samples collected from 306 GC patients. Correlations between NPC1 expression, clinical characteristics, and patient prognosis were analyzed. NPC1 mRNA expression was elevated in GC tissues compared to normal tissues (P < 0.05) and significantly associated with poorer prognosis. In our cohort of 306 patients, NPC1 exhibited significant upregulation in GC versus adjacent normal tissues (P = 0.031). High NPC1 expression correlated with adverse clinical characteristics, including lymph node metastasis, distant metastasis, and advanced TNM stage (all P < 0.05). Patients with high NPC1 expression experienced notably shorter overall survival (P < 0.001), particularly in stages III and IV (P = 0.003). Multivariate Cox regression analysis identified high NPC1 expression as an independent prognostic factor for GC patients (HR 1.57, 95% CI 1.14-2.18, P = 0.006). Lastly, an optimized nomogram incorporating NPC1, tumor size, and TNM stage was constructed. NPC1 expression is upregulated in GC and serves as a pivotal prognostic factor for adverse outcomes in GC patients.

Functional Characterization of the Niemann-Pick C2 Protein BdioNPC2b in the Parasitic Wasp Baryscapus dioryctriae (Chalcidodea: Eulophidae).

Reverse chemical ecology has been widely applied for the functional characterization of olfactory proteins in various arthropods, but few related studies have focused on parasitic wasps. Here, the odorant carrier Niemann-Pick C2 protein of Baryscapus dioryctriae (BdioNPC2b) was studied in vitro and in vivo. Ligand binding analysis revealed that BdioNPC2b most strongly bound to 2-butyl-2-octenal and which compound could elicit an EAG response and attracted B. dioryctriae adults. Moreover, this odorant attractant significantly improved the reproductive efficiency of B. dioryctriae compared to that of the control. Then, the relationship between BdioNPC2b and 2-butyl-2-octenal was validated by RNAi, and site-directed mutagenesis revealed the involvement of three key residues of BdioNPC2b in binding to 2-butyl-2-octenal through hydrogen bonding. Our findings provide not only a deeper understanding of the olfactory function of NPC2 in wasps but also useful information for improving the performance of the parasitoid B. dioryctriae as a biological control agent.

Mitochondrial dysfunction in NPC1-deficiency is not rescued by drugs targeting the glucosylceramidase GBA2 and the cholesterol-binding proteins TSPO and StARD1.

Niemann-Pick type C disease (NPCD) is a rare neurodegenerative disorder most commonly caused by mutations in the lysosomal protein Niemann-Pick C1 (NPC1), which is implicated in cholesterol export. Mitochondrial insufficiency forms a significant feature of the pathology of this disease, yet studies attempting to address this are rare. The working hypothesis is that mitochondria become overloaded with cholesterol which renders them dysfunctional. We examined two potential protein targets-translocator protein (TSPO) and steroidogenic acute regulatory protein D1 (StARD1)-which are implicated in cholesterol transport to mitochondria, in addition to glucocerbrosidase 2 (GBA2), the target of miglustat, which is currently the only approved treatment for NPCD. However, inhibiting these proteins did not correct the mitochondrial defect in NPC1-deficient cells.

Cyclodextrin-Mediated Cholesterol Depletion Induces Adiponectin Secretion in 3T3-L1 Adipocytes.

Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.

NPC1 plays a role in the trafficking of specific cargo to melanosomes

Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.

Structural and Energetic Basis for Differential Binding of Ebola and Marburg Virus Glycoproteins to a Bat-Derived Niemann-Pick C1 Protein.

Our previous study demonstrated that the fruit bat (Yaeyama flying fox)-derived cell line FBKT1 showed preferential susceptibility to Ebola virus (EBOV), whereas the human cell line HEK293T was similarly susceptible to EBOV and Marburg virus (MARV). This was due to 3 amino acid differences of the endosomal receptor Niemann-Pick C1 (NPC1) between FBKT1 and HEK293T (ie, TET and SGA, respectively, at positions 425-427), as well as 2 amino acid differences at positions 87 and 142 of the viral glycoprotein (GP) between EBOV and MARV. To understand the contribution of these amino acid differences to interactions between NPC1 and GP, we performed molecular dynamics simulations and binding free energy calculations. The average binding free energies of human NPC1 (hNPC1) and its mutant having TET at positions 425-427 (hNPC1/TET) were similar for the interaction with EBOV GP. In contrast, hNPC1/TET had a weaker interaction with MARV GP than wild-type hNPC1. As expected, substitutions of amino acid residues at 87 or 142 in EBOV and MARV GPs converted the binding affinity to hNPC1/TET. Our data provide structural and energetic insights for understanding potential differences in the GP-NPC1 interaction, which could influence the host tropism of EBOV and MARV.

Identification of novel compound heterozygous mutation in Niemann-Pick disease type C gene (P14-11.006)

<h3>Objective:</h3> To report a novel compound heterozygous mutation in two siblings with Niemann-Pick disease type C who displayed distinct phenotypic presentation <h3>Background:</h3> Niemann–Pick disease type C (NPC) is a rare autosomal recessive lysosomal storage disease with heterogenous neurological and systemic manifestations. The vast majority of cases (95%) are caused by mutations in the Niemann–Pick C1 protein (NPC1), whereas the reminder of cases are attributed to mutations in Niemann–Pick C2 protein (NPC2). <h3>Design/Methods:</h3> Case report <h3>Results:</h3> <h3>Case 1:</h3> A 57-year-old female presented with a 5-year history of progressive slowness, poor coordination, and cognitive decline. This was followed by generalized stiffness and recurrent ballistic movements within two years of onset. Her poor postural stability lead to frequent falls. Initial neurological evaluation revealed severe dysarthric speech, moderate vertical eye movement limitation, spasticity and exaggerated deep tendon reflexes, along with dystonic movements. She progressed rapidly, developing generalized dystonia which made her bedbound. <h3>Case 2:</h3> the young sibling of case 1, presented at the age of 56 with 8-year history of poor balance. Symptoms were slowly progressive, noted initially with changes in his balance, slurred speech, and poor coordination. He was also noticed to have mild cognitive changes. Examination revealed cerebellar dysarthria, with failure to generate saccades from primary position downwards. Deep tendon reflexes were brisk, with no evidence of muscle wasting, or spasticity. He showed signs of mild axial and appendicular ataxia. The progression was slow, and he maintained his mobility without requiring walking aids. Molecular genetic testing revealed compound heterozygous mutation in the NPC1 gene; c.180G&gt;T (p.Gln60His) and c.3173C&gt;A (p.Ala1058Asp). All biochemical results were within normal range. Brain MRI showed generalized cerebral and cerebellar volume loss few years into the onset of symptoms in both patients. <h3>Conclusions:</h3> Our report highlights the high intrafamilial heterogeneity and poor genotype-phenotype correlation in the case of Niemann–Pick disease type C. <b>Disclosure:</b> Dr. Abusrair has nothing to disclose. Dr. Amorelli has nothing to disclose. Justyna Sarna has nothing to disclose.

Niemann-Pick C1 protein regulates platelet membrane–associated calcium ion signaling in thrombo-occlusive diseases in mice

BackgroundPathophysiologic platelet activation leads to thrombo-occlusive diseases such as myocardial infarction or ischemic stroke. Niemann-Pick C1 protein (NPC1) is involved in the regulation of lysosomal lipid trafficking and calcium ion (Ca2+) signaling, and its genetic mutation causes a lysosomal storage disorder. Lipids and Ca2+ are key players in the complex orchestration of platelet activation. ObjectivesThe present study aimed to determine the impact of NPC1 on Ca2+ mobilization during platelet activation in thrombo-occlusive diseases. MethodsUsing MK/platelet-specific knockout mice of Npc1 (Npc1Pf4∆/Pf4∆), ex vivo and in vitro approaches as well as in vivo models of thrombosis, we investigated the effect of Npc1 on platelet function and thrombus formation. ResultsWe showed that Npc1Pf4∆/Pf4∆ platelets display increased sphingosine levels and a locally impaired membrane-associated and SERCA3-dependent Ca2+ mobilisation compared to platelets from wildtype littermates (Npc1lox/lox). Further, we observed decreased platelet. ConclusionOur findings highlight that NPC1 regulates membrane-associated and SERCA3-dependent Ca2+ mobilization during platelet activation and that MK/platelet-specific ablation of Npc1 protects against experimental models of arterial thrombosis and myocardial or cerebral ischemia/reperfusion injury.

Rab7a-mTORC1 signaling-mediated cholesterol trafficking from the lysosome to mitochondria ameliorates hepatic lipotoxicity induced by aflatoxin B1 exposure

Aflatoxin B1 (AFB1) is a common contaminant in many foodstuffs and is considered a public health concern worldwide due to its hepatotoxicity caused by lipid metabolism disorders. However, the molecular mechanism underlying AFB1-induced lipotoxicity-dependent liver injury via regulating cholesterol metabolism remains unclear. We established a cholesterol trafficking disorder-mediated hepatic lipotoxicity model with AFB1 mixture exposure in vitro (HepaRG and HepG2 cells, 1.6 μM for 36 h) and in vivo (C57BL/6 mice, 3 mg kg−1, i.g., every other day for 6 weeks). In vitro, the interaction between lysosomal Niemann-Pick type C1 (NPC1) protein and mitochondrial translocator protein (TSPO) regulated lipotoxicity induced by AFB1 mixture exposure, including lysosomal membrane permeabilization and mitochondria-dependent necroptosis. Moreover, the downregulation of lysosomal Ras-associated protein 7a (Rab7a) enhanced the mammalian target of rapamycin complex 1 (mTORC1)-mediated disorders of cholesterol trafficking from the lysosome to mitochondria. Furthermore, cholesterol trafficking disorder-mediated hepatic lipotoxicity induced by the low-dose level of AFB1 exposure was relieved by genetic or pharmaceutic activation of Rab7a to inhibit mTORC1 in vitro and ex vivo. In vivo, mTORC1 inhibitor (Torin1, 4 mg kg−1, i.p., every other day for 3 weeks) alleviated the cholesterol trafficking disorder-mediated hepatic lipotoxicity via upregulating the molecular machinery of lysosomes and mitochondria contact mediated by NPC1 and TSPO interaction in the low dose of AFB1 exposure. Altogether, our data suggested a novel mechanism that lysosomal Rab7a-mTORC1 signaling determined the cholesterol trafficking regulated by NPC1-TSPO from the lysosome to mitochondria, which promoted hepatic lipotoxicity via lysosomal quality control and mitochondria-dependent necroptosis signaling pathways in chemical mixture exposure.

Purification and structure of luminal domain C of human Niemann-Pick C1 protein.

Niemann-Pick C1 protein (NPC1) is a membrane protein that primarily resides in late endosomes and lysosomes, and plays an important role in cholesterol homeostasis in the cell. The second luminal domain of NPC1 (NPC1-C) serves as the intracellular receptor for Ebola and Marburg viruses. Here, the recombinant production of nonglycosylated and glycosylated NPC1-C and a new crystal form of the nonglycosylated protein are reported. The crystals belonged to space group P21 and diffracted to 2.3 Å resolution. The structure is similar to other reported structures of NPC1-C, with differences observed in the protruding loops when compared with NPC1-C in complex with Ebola virus glycoprotein or NPC2.

Abstract 1502: Misrouting of Niemann-Pick C1 protein mediates cholesterol induced mTORC1-activation contributing to pathogenesis of CLN1-disease

Abstract 1502: Misrouting of Niemann-Pick C1 protein mediates cholesterol induced mTORC1-activation contributing to pathogenesis of CLN1-disease

Proteomic analysis of s-acylated proteins in human retinal pigment epithelial cells and the role of palmitoylation of Niemann-Pick type C1 protein in cholesterol transport.

Palmitoylation is a dynamic process that regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in the visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 55 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant enrichment of palmitoylated proteins in cell-matrix adhesion, cell-cell recognition, protein cellular localization, and translation, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900, and 816) of Niemann-Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys799 and 800 caused significant cholesterol accumulation in the late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. However, the mutation did not alter the cellular localization of the protein. Conclusion: PRE cells have an abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol export, but not the intracellular localization of NPC1.

Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria

Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.

Ligand-based design of peptide entry inhibitors targeting the endosomal receptor binding site of filoviruses

Filoviruses enter cells through macropinocytosis and trafficking into the endosomes in which they bind to the receptor Niemann-Pick C1 protein (NPC1) for membrane fusion and entry into the cytoplasm. The endosomal receptor-binding is critical step for filovirus entry. Designing inhibitors to block receptor binding will prevent viral entry. Using available binding structural information from the co-crystal structures of the viral GP with the receptor NPC1 or with monoclonal antibodies, we have conducted structure-based design of peptide inhibitors to target the receptor binding site (RBS). The designed peptides were tested for their inhibition activity against pseudo-typed or replication-competent viruses in a cell-based assay. The results indicate that these peptides exhibited strong activities against both Ebola and Marburg virus infection. It is expected that these peptides can be further developed for therapeutic use to treat filovirus infection and combat the outbreaks.

Identification and expression of potential olfactory-related genes related to Niemann-Pick C2 protein and ionotropic receptors in Haemaphysalis longicornis.

Studies have shown that the main pathway for tick host localization and perception of mating information may be chemosensory. However, chemical communication in ticks is poorly understood, especially in those other than the Ixodes ticks. Niemann-Pick C2 (NPC2) protein and ionotropic receptors (IRs) are considered to be closely related to the perception of infochemicals in arthropods. Through bioinformatic analysis, eight NPC2 and four IR candidate genes were identified through screening and identification of the transcriptome sequencing database of Haemaphysalis longicornis. Phylogenetic tree analysis indicated that H. longicornis possesses similar homology to the genus Ixodes. A comparison of the expression of NPC2 and IR in tick forelegs (first pair of legs), hind legs (fourth pair of legs), and capitula using RT-PCR revealed that, barring HlonNPC2-8, 11 candidate genes were highly expressed in the foreleg and capitulum, which are the main sensory organs of ticks. They were also expressed in the hind legs, except for six genes that were not expressed in the males. RT-qPCR analysis showed upregulation and higher relative expression of HlonNPC2-1, HlonNPC2-3, HlonNPC2-6, and HlonNPC2-8 when stimulated by ammonium hydroxide, whereas the others were downregulated and demonstrated lower relative expression. These results further support the putative role of NPC2s as a new odorant carrier in ticks and present 12 promising candidate genes for understanding tick olfactory communication, enriching the data on these genes, especially outside the genus Ixodes.

The point mutation of the cholesterol trafficking membrane protein NPC1 may affect its proper function in more than a single step: Molecular dynamics simulation study

The Niemann-Pick type C1 (NPC1) protein is one of the key players of cholesterol trafficking from the lysosome and its function is closely coupled with the Niemann-Pick type C2 (NPC2) protein. The dysfunction of one of these proteins can cause problems in the overall cholesterol homeostasis and leads to a disease, which is called the Niemann-Pick type C (NPC) disease. The parts of the cholesterol transport mechanism by NPC1 have begun to recently emerge, especially after the full-length NPC1 structure was determined from a cryo-EM study. However, many details about the overall cholesterol trafficking process by NPC1 still remain to be elucidated. Notably, the NPC1 could act as one of the target proteins for the control of infectious diseases due to its role as the virus entry point into the cells as well as for cancer treatment due to the inhibitory effect of tumor growth. A mutation of NPC1 can leads to dysfunctions and understanding this process can provide valuable insights into the mechanisms of the corresponding protein and the therapeutic strategies against the disease that are caused by the mutation. It has been found that patients with the point mutation R518W (or R518Q) on the NPC1 show the accumulation of lipids within the lysosomal lumen. In this paper, we report how the corresponding mutation can affect the cholesterol transport process by NPC1 in the different stages by the molecular dynamics simulations. The simulation results show that the point mutation intervenes at least at two different steps during the cholesterol transport by NPC1 and NPC2 in combination, which includes the association step of NPC2 with the NPC1, the cholesterol transfer step from NPC2 to NPC1-NTD while the cholesterol passage within the NPC1 via a channel is relatively unaffected by R518W mutation. The detailed analysis of the resulting simulation trajectories reveals the important structural features that are essential for the proper functioning of the NPC1 for the cholesterol transport, and it shows how the overall structure, which thereby includes the function, can be affected by a single mutation.