【作者】 高原；

【导师】 季维克；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2021， 博士

【摘要】 背景:内质网(endoplasmic reticulum,ER)和晚期内吞体/溶酶体(late endosome/lysosomes,LE/lys)之间的膜接触位点(membrane contact sites,MCSs)是多种重要细胞活动(内吞体成熟、运输以及细胞内脂质和离子稳态维持)的调控枢纽,其异常与神经系统疾病的发生密切相关。然而,关于这些MCSs的组成、调控和功能目前仍不清楚。目的:鉴定未知的ER-LE/lys MCSs关键蛋白,研究关键新分子在ER-LE/lys MCSs的作用及细胞生物学功能。方法:我们通过免疫共沉淀结合质谱分析,鉴定了与已知的ER-LE/lys MCSs系链蛋白Protrudin相互作用的蛋白。其中,一个含有SMP结构域的蛋白PDZD8,由于其潜在的脂质转移活性,引起了我们的极大兴趣。我们首先通过活细胞共聚焦成像,研究了PDZD8与Protrudin的细胞定位,我们发现PDZD8与Protrudin存在较好的共定位,提示PDZD8是定位于ER-LE/lys MCSs的蛋白。我们进一步通过Lattice-SIM超高分辨率成像验证了PDZD8相对于ER-LE/lys MCSs和ER-线粒体膜接触位点的定位。根据蛋白质结构域分析,我们构建了PDZD8的多个截断突变体。通过活细胞共聚焦成像,我们鉴定了PDZD8靶向ER和LE/lys的区域。我们利用质谱鉴定与PDZD8相互作用的LE/lys膜蛋白,通过活细胞共聚焦成像和pull-down实验发现PDZD8-C1-CC区域直接结合活性形式的小GTPase Rab7,从而靶向LE/lys。接下来我们系统研究了PDZD8的脂质结合和转运能力。我们通过质谱分析,结合体外脂质结合实验,研究了PDZD8的脂质结合能力。进一步,通过基于荧光共振能量转移的体外脂质转移实验,检测了SMP结构域在脂质体膜间转移脂质的能力。功能上,我们通过PDZD8敲降实验,研究了PDZD8对于LE/lys的分布影响,进一步利用NGF诱导的PC12细胞分化来研究PDZD8是否在神经元突起生长中起重要作用。结果:我们发现PDZD8是直接介导ER-LE/lys MCSs形成的系链蛋白,并与ERLE/lys MCSs的系链蛋白Protrudin的TM结构域相互作用。PDZD8通过其N端的TM结构域锚定在ER膜上,通过其C端的C1-CC区域直接与Rab7结合靶向LE/lys。PDZD8介导的ER-LE/lys MCSs受Rab7活性的调控。PDZD8的SMP结构域可以在细胞内和体外结合甘油磷脂和神经酰胺,并在体外介导脂质在脂质体膜间转运。在功能上,PDZD8对于LE/lys的胞内正向运输和神经元突起的生长至关重要。同时,我们发现PDZD8的这些细胞生物学功能依赖于PDZD8的SMP结构域的脂质转移活性。结论:PDZD8能够直接介导ER-LE/lys MCSs的形成。PDZD8的SMP结构域结合并转运甘油磷脂和神经酰胺。PDZD8在LE/lys的胞内正向运输和神经元突起的生长过程中起到重要作用。PDZD8的SMP结构域介导的脂质转移是LE/lys转运和神经元突起生长的关键调控因子。更多还原

【Abstract】 Backgroud: Membrane contact sites between endoplasmic reticulum(ER)and late endosomes/lysosomes(LE/lys)are emerging as critical hubs for diverse cellular events(endosomal maturation and transport,maintenance of intracellular lipid and ion homeostasis),and changes in their extents are linked to severe neurological diseases.However,molecular composition,regulation and coordination of tether protein complexes between these membrane contacts are still elusive.Objective: To identify key factors at ER-LE/lys MCSs tether protein,and further elucidate the roles and mechanisms of the key factor at ER-LE/lys MCSs,and eventually explore the cellular functions of the ER-LE/lys MCSs mediated by the key factor.Methods: To identify unknown proteins that may interact with Protrudin,a known ER-LE/lys MCSs tether protein,we performed GFP-trap assays followed by mass spectrometry(MS)analysis.A SMP domain containing protein named PDZ domain containing protein 8(PDZD8)strongly interested us due to its potential lipid transfer activities.Therefore we explored cellular localizations of PDZD8 and Protrudin by live-cell confocal microscopy.We further examined the localization of PDZD8 relative to LE/lys,ER and mitochondria by super-resolution Lattice-SIM microscopy.Based on protein domain analysis,we constructed multiple truncated mutants of PDZD8,and confirmed the regions of PDZD8 targeting ER and LE/lys,respectively,by live-cell confocal microscopy.We identified PDZD8-interacting proteins by GFP-trap assays followed by MS analysis,and found that PDZD8-C1-CC region targeted LE/lys by directly binding Rab7.We next tested the possibility that PDZD8 might directly bind and transport lipids.We confirmed the lipid binding ability of PDZD8 by mass spectrometry(MS)analysis and lipid binding assay in vitro.We next employed a fluorescence resonance energy transfer(FRET)–based lipid transfer assay to examine whether the SMP domain can transfer lipids between membranes in vitro.We further investigated whether PDZD8 played important roles in the process of LE/lys trafficking and neurite outgrowth using Neuronal growth factor(NGF)stimulated PC12 differentiation PC12 cells by confocal microscopy.Results: We showed that PDZD8 was a tether at ER-LE/lys MCSs and interacted with the TM domain of Protrudin at such MCSs.PDZD8 anchored into the ER membrane with its N terminal TM domain while the C-terminal C1-CC region recognized LE/lys membrane through directly binding to Rab7.PDZD8 mediated ER-LE/lys MCSs were regulated by Rab7 activities.The SMP domain bound to glycerophospholipids and ceramides and mediate lipids transport between membranes in vitro.Functionally,PDZD8 mediated proper cellular positioning of LE/lys and promoted neurite outgrowth,which was lipid transfer activity dependent.Conclusion: PDZD8 mediated ER-LE/lys MCSs and its SMP domain bound to glycerophospholipids and ceramides and mediate lipids transport between membranes in vitro.PDZD8 functions as a tether at ER-LE/lys MCSs and is required for LE/lys positioning and neurite extensions through lipid transfer activities mediated by SMP domain.更多还原