NPC1L1:固醇脂质吸收的关键蛋白质

NPC1L1是最近发现的一种与NPC1同源的蛋白质。在体内的分布有物种差异性,其亚细胞定位存在很大争议。近些年发现NPC1L1在固醇类脂质代谢途径中起着重要作用,是肠道吸收固醇类脂质尤其是胆固醇的关键蛋白质,这项新发现使得人们对固醇类脂质的吸收机制有了了解。高胆固醇血症是心血管系统疾病的一个高危因子,因此,对NPC1L1的研究具有重大的实际意义,正逐渐成为研究的热点。     

ABCA1与NPC1在细胞内胆固醇转运中的作用

腺苷三磷酸结合盒转运蛋白A1（ATP-binding cassette transporter A1,ABCA1）是血浆高密度脂蛋白（high-density lipoprotein,HDL）颗粒形成之初的限速步骤。ABCA1通过膜泡运输脂质至细胞表面的HDL载脂蛋白的作用机制尚未完全阐明。C型尼曼-匹克病（Niemann-Pick disease type C,NPC）主要由NPC1基因突变引起,NPC1蛋白能促进胆固醇和其他脂质从晚期胞内体/溶酶体流入其他细胞结构。ABCA1和NPC1相互作用保持细胞内脂质平衡,与Tangier病和N C P病等病理过程密切相关。     

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

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

Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian–dinoflagellate symbiosis

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.     

The Cytosolic Adaptor AP‐1A Is Essential for the Trafficking and Function of Niemann‐Pick Type C Proteins

Niemann‐Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over‐accumulation of low‐density lipoprotein‐derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three‐hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP‐1 via its acidic/di‐leucine motif. Consequently, a nonfunctional AP‐1A cytosolic complex resulted in a typical NPC‐like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ‐cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP‐1A is essential for the lysosomal targeting and function of NPC1 and NPC2.     

Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans

The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann–Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.     

用PO4细胞为靶细胞检测人血清中Epstein-Barr病毒IgA/MA抗体以诊断鼻咽癌

近年来,常用未经丙酮固定的、由丁酸和巴豆油激活的B95-8细胞或P3HR-1细胞为靶细胞,检测人血清中EB病毒IgA/MA抗体以早期诊断鼻咽癌,效果良好。但由于B95-8细胞含有多种EB病毒抗原,不能用丙酮固定,需多次离心沉淀,在浮悬状态下检测,技术比较复杂。     

Targeted inhibition of the EGFR pathways enhances Zn‐BC‐AM PDT‐induced apoptosis in well‐differentiated nasopharyngeal carcinoma cells

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn‐BC‐AM PDT with an EGFR inhibitor AG1478 were investigated. Well‐differentiated NPC HK‐1 cells were subjected to PDT with 1 µM of Zn‐BC‐AM and were irradiated at a light dose of 1 J/cm² in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK‐1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub‐lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn‐BC‐AM PDT in HK‐1 cells. Pre‐incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn‐BC‐AM PDT‐induced formation of apoptotic cells. The efficacy of Zn‐BC‐AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn‐BC‐AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356–1363, 2009. © 2009 Wiley‐Liss, Inc.     

Sterol-binding proteins and endosomal cholesterol transport

Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis. A large number of studies have focused on the removal of endosomal cholesterol, since its accumulation leads to devastating human diseases. Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport. In particular, endosome/lysosome-localized or -associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner. Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.     

NP c 1L l 在高脂血症和动脉粥样硬化中的表达分析

目的：研究NPC1L1（Niemann-Pick C1 Like 1）mRNA在单纯高脂血症大鼠和动脉粥样硬化大鼠小肠组织中的表达与差异,探讨其与脂质代谢和动脉粥样硬化之间的关系。方法：通过半定量RT-PCR方法分别检测正常普食组、单纯高脂饲养组和动脉粥样大鼠组小肠组织中NPC1L1 mRNA的表达差异。结果：三个组别大鼠小肠组织中均存在NPC1L2 mRNA,单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达明显高于正常对照大鼠（P〈0．01）;单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达之间无明显差异（P〉0．05）。结论：血脂代谢紊乱与小肠组织中NPC1L1的高表达有关,NPC1L1可能参与了血脂紊乱的病理生理过程;NPC1L1与促成动脉粥样硬化的发生无明显相关性。     

细胞内胆固醇运输

作为生物膜的重要成分,细胞内不同膜上胆固醇含量的高低直接影响生物膜的生物物理特性和细胞信号的传递,与细胞正常的生理功能密切相关。外源内吞的胆固醇和内源合成的胆固醇通过囊泡介导和非囊泡介导的胆固醇运输途径在不同细胞膜之间转运,从而维持了不同细胞器上胆固醇的浓度梯度。一系列胆固醇结合和转运蛋白在细胞内胆固醇的运输中发挥了重要作用。本文旨在总结细胞内胆固醇运输途径与参与胆固醇运输的重要分子及相关作用机制。     

Lipids on Trial: The Search for the Offending Metabolite in Niemann‐Pick type C Disease

Niemann‐Pick disease type C is a complex lysosomal storage disorder caused by mutations in either the NPC1 or NPC2 genes that is characterized at the cellular level by the storage of multiple lipids, defective lysosomal calcium homeostasis and unique trafficking defects. We review the potential role of each of the individual storage lipids in initiating the pathogenic cascade and propose a model of NPC1 and NPC2 function based on the current knowledge     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

Are Niemann‐Pick type C proteins key players in cnidarian–dinoflagellate endosymbioses?

The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host–symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol‐trafficking proteins Niemann‐Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene in contrast to the two genes (NPC1 and NPC1L1) present in vertebrate genomes. While only one NPC2 gene is present in many metazoans, this gene has been duplicated in cnidarians, and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2‐d) was upregulated in symbiotic relative to aposymbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont‐containing tissue) than in the epidermis. We performed immunolabelling experiments on tentacle cross sections and demonstrated that the AvNPC2‐d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2‐d gene expression was strongly downregulated during stress. These data suggest that AvNPC2‐d is involved in both the stability and dysfunction of cnidarian–dinoflagellate symbioses.     

The role of Exosomes in the Pathogenesis of Nasopharyngeal Carcinoma and the involved Clinical Application

Exosomes are nanoscale membrane vesicles, which carry biologically active substances of their cell of origin and play an important role in signal transduction and intercellular communication. At present, exosomes have been identified as a promising non-invasive liquid biopsy biomarker in the tissues and circulating blood of nasopharyngeal carcinoma (NPC) and found to participate in regulating pathophysiological process of the tumor. We here review recent insights gained into the molecular mechanisms of exosome-induced cell growth, angiogenesis, metastasis, immunosuppression, radiation resistance and chemotherapy resistance in the development and progression of NPC, as well as the clinical application of exosomes as diagnostic biomarkers and therapeutic agents. We also discuss the limitations and challenges in exosome application. We hope this review may provide some references for the use of exosomes in clinical intervention.     

Epstein-Barr病毒和鼻咽癌血清学诊断

EB病毒与鼻咽癌关系密切。 EB病毒抗原可用于鼻咽癌早期诊断以提高鼻咽癌病人存活率。本文对 EB病毒生活周期及其抗原系统 ,EB病毒与鼻咽癌之间关系及应用 EB病毒抗原进行鼻咽癌血清学诊断的进展作一概括介绍     

c-Myc-induced circ-NOTCH1 promotes aggressive phenotypes of nasopharyngeal carcinoma cells by regulating the miR-34c-5p/c-Myc axis

Nasopharyngeal carcinoma (NPC) is the subclass of head and neck cancer with the highest incidence among otolaryngology malignancies. A growing amount of evidence has proven that circular RNAs (circRNAs) play key roles in the progression of multiple cancers. It has been reported that circ-NOTCH1 is a novel circRNA and functions as an oncogene in gastric cancer, while the regulatory mechanism of circ-NOTCH1 in NPC remains unknown. In the present research, our findings revealed that circ-NOTCH1 was overexpressed in NPC tissues and cells. Circ-NOTCH1 knockdown suppressed NPC cell proliferation, invasion, and migration. Subsequently, we discovered that c-Myc can activate circ-NOTCH1 by binding to the NOTCH1 promoter. c-Myc functioned as a tumor promoter in NPC cells. Mechanistically, circ-NOTCH1 served as a competitive endogenous RNA to modulate c-Myc expression by sponging miR-34c-5p. Additionally, overexpression of c-Myc reversed the circ-NOTCH1 knockdown-mediated inhibition of NPC cellular progression. Overall, this study suggested that c-Myc-induced circ-NOTCH1 promoted malignant phenotypes of NPC cells by regulating the miR-34c-5p/c-Myc axis.