Computational screening of disease associated mutations on NPC1 gene and its structural consequence in Niemann-Pick type-C1

Niemann-Pick disease type C1 （NPC1）, caused by mutations of NPC1 gene, is an inherited lysosomal lipid storage disorder. Loss of functional NPC1 causes the accumulation of free cholesterol （FC） in endocytic organelles that comprised the characteristics of late endosomes and/or lysosomes. In this study we analyzed the pathogenic effect of 103 nsSNPs reported in NPC1 using computational methods. Rl186C, S940L, R958Q and I1061T mutations were predicted as most deleterious and disease associated with NPC1 using SIFT, Polyphen 2.0, PANTHER, PhD-SNP, Pmut and MUTPred tools which were also endorsed with previous in vivo experimental studies. To understand the atomic arrangement in 3D space, the native and disease associated mutant （Rl186C, S940L, R958Q and I1061T） structures were modeled. Quantitative structural and flexibility analysis was conceded to observe the structural consequence of prioritized disease associated mutations （R1186C, S940L, R958Q and I1061T）. Accessible surface area （ASA）, free folding energy （FFE） and hydrogen bond （NH bond） showed more flexibility in 3D space in mutant structures. Based on the quantitative assessment and flexibility analysis of NPC1 variants, I1061T showed the most deleterious effect. Our analysis provides a clear clue to wet laboratory scientists to understand the structural and functional effect of NPCI gene upon mutation.     

溶酶体贮积症的研究进展

溶酶体贮积症是一种罕见的遗传缺陷疾病,溶酶体内未酶解的大分子累积,最终导致细胞功能障碍和临床异常情况。许多溶酶体底物在细胞结构和功能上都有关键的作用,因此溶酶体功能失常的影响非常广泛,如神经受累、间质受累、网状内皮组织受累及胎儿水肿。治疗方法主要有骨髓移植、酶替代疗法、底物减少治疗、基因治疗和分子伴侣治疗。利用转基因及其他一些前沿技术,将有可能彻底根除这些长期困扰人类的溶酶体贮积症。     

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2^−/− and npc1^−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.     

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C

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Structure–activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure–activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

FrzA基因转导干预缺血性心力衰竭小鼠心肌Wnt信号通路的研究

目的:研究重组9型腺相关病毒(recombinant adeno-associated virus serotype 9,rAAV9)携带FrzA基因转导干预缺血性心衰小鼠心肌Wnt信号通路的可行性,为基因治疗心力衰竭提供新的思路.方法:选择3月龄雄性C57BL/6J小鼠共130只,随机分为空白组(n=10),心衰组(n=40),心衰+空病毒(rAAV9-GFP)注射组(n=40),心衰+rAAV9-FrzA组(n=40),采用结扎左冠状动脉定量控制心梗面积,于术后2周行心脏超声评各组心功能变化,再经尾静脉注射已稀释好的病毒,28d后处死小鼠取心脏标本,RT-PCR检测心肌目的基因FrzA以及Dvl-1,β-catenin的表达;Western blot检测心肌Wnt信号通路关键分子Dvl-1,GSK3β,p-GSK3β,β-catenin的表达.结果:与空白组相比,成功建立心衰模型后小鼠心功能均不同程度降低(P＜0.05);FrzA组与心衰组相比,心功能明显改善(P＜0.05);经尾静脉注射可成功将rAAV9-FrzA导入小鼠体内,并且目的基因FrzA在心肌组织中高表达(P＜0.05);心衰小鼠心肌中Wnt信号通路关键分子Dvl-1,p-GSK3β,β-catenin的表达显著升高(P＜0.05),FrzA转导后小鼠心肌中Wnt信号通路中关键分子Dvl-1,p-GSK3β,β-catenin表达均降低(P＜0.05)结论:利用rAAV9-FrzA转导缺血性心衰小鼠可以有效的干预心肌Wnt信号通路,抑制其活性,为基因治疗缺血性心衰提供了新的思路.     

重组腺相关病毒生产方法研究进展

重组腺相关病毒(rAAV)作为基因治疗的载体,具有感染范围广、能在宿主细胞中长久稳定表达外源基因和非致病性等优点,因此在基因治疗领域倍受青睐。rAAV的大规模制备技术一直是限制其临床广泛应用的瓶颈。近年来,有许多改进rAAV制备工艺的尝试。我们在介绍rAAV载体制备所需各种元件的基础上,对这些方法进行了简要综述。     

2型腺相关病毒载体介导的apoAⅠ和SR-BⅠ双基因表达对动脉硬化模型鼠保护效应的研究

重组腺相关病毒载体(AAV)具有很多安全方面的特性,有利于其在动脉硬化方面的治疗研究.尽管如此,传统的介导单个基因的治疗效果并不是很理想,这都归因于动脉硬化疾病的发生是由于多种基因的缺陷而不是单单某一特定基因.为了克服这个问题,尝试了重组腺相关病毒载体介导的双基因对动脉硬化的治疗研究.实验大鼠分为动脉硬化模型鼠和正常饮食组(即正常对照组),动脉硬化组大鼠被随机分为3组,分别进行AAV-apoAⅠ/SR-BⅠ,AAV-apoAⅠ,与AAV-GFP尾静脉注射,同时正常饮食组尾静脉注射PBS作为对照.其中目的基因mRNA检测采用RT-PCR方法,蛋白质表达的检测采用Western blotting和ELISA.由饮食诱导的动脉硬化和高胆固醇的大鼠模型在尾静脉注射后8周进行冰冻切片的荧光检测.重组AAV载体显示出较强的表达活性.尾静脉注射治疗8周后,AAV-apoAⅠ/SR-BⅠ与AAV-apoAⅠ治疗组血浆总胆固醇和低密度脂蛋白胆固醇浓度与AAV-GFP治疗组相比有了明显下降(P<0.05),高密度脂蛋白胆固醇浓度各组之间没有明显差异,彩色多普勒超声检测发现,AAV-apoAⅠ/SR-BⅠ与AAV-apoAⅠ治疗组的腹主动脉的内中膜厚度相对于AAV-GFP治疗组有了明显下降(P<0.05),血清hs-CRP和SOD的水平有了明显上升(P<0.01),血清MDA的水平有了明显的下降(P<0.01).同时也检测了动脉硬化相关基因mRNA水平的表达.结果显示,rAAV-hapoAⅠ-IRES-hSR-BⅠ治疗后可能是通过抑制NF-κB的活性发挥抗炎作用减少炎症因子的释放,增加动脉硬化板块的稳定性以及降低血浆胆固醇含量的.总之,利用2型腺相关病毒载体介导的基因转移过度表达人载脂蛋白AⅠ和SR-BⅠ可能对饮食诱发大鼠高胆固醇血症和动脉硬化的产生有利影响.这些结果可能为动脉粥样硬化基因治疗提供了一种新的研究思路.     

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2^−/− fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.