L-periaxin与Ezrin的N端FERM结构域结合依赖其第3段核定位信号序列北大核心CSCD

L-periaxin是外周神经系统特异表达的骨架蛋白之一,占外周神经系统髓鞘总蛋白质的16%,参与髓鞘形成和维护。埃兹蛋白(Ezrin)属于Ezrin-Radxin-Moesin(ERM)蛋白质家族,与细胞黏附、迁移、生存以及肿瘤的发生、发展相关。作者前期研究证实,L-periaxin与Ezrin通过"头对头,尾对尾"的模式相互结合。本文通过双分子荧光互补、免疫共沉淀、GST pull down、荧光共定位、海肾荧光素酶互补、荧光光谱以及分子对接等方法,揭示L-periaxin的核定位信号区(nuclear location signal,NLS)与蛋白质Ezrin的FERM(Ezrin Radixin Moesin)结构域的"头对头"相互结合,依赖Lperiaxin第3段核定位信号序列NLS3与Ezrin的FERM结构域的F3亚结构域。本结果为进一步理解Ezrin在髓鞘化过程中的作用,以及阐明调节L-periaxin蛋白功能的信号通路奠定基础。     

L-periaxin与Ezrin的N端FERM结构域结合依赖其第3段核定位信号序列

L-periaxin是外周神经系统特异表达的骨架蛋白之一,占外周神经系统髓鞘总蛋白质的16%,参与髓鞘形成和维护。埃兹蛋白(Ezrin)属于Ezrin-Radxin-Moesin(ERM)蛋白质家族,与细胞黏附、迁移、生存以及肿瘤的发生、发展相关。作者前期研究证实,L-periaxin与Ezrin通过"头对头,尾对尾"的模式相互结合。本文通过双分子荧光互补、免疫共沉淀、GST pull down、荧光共定位、海肾荧光素酶互补、荧光光谱以及分子对接等方法,揭示L-periaxin的核定位信号区(nuclear location signal,NLS)与蛋白质Ezrin的FERM(Ezrin Radixin Moesin)结构域的"头对头"相互结合,依赖Lperiaxin第3段核定位信号序列NLS3与Ezrin的FERM结构域的F3亚结构域。本结果为进一步理解Ezrin在髓鞘化过程中的作用,以及阐明调节L-periaxin蛋白功能的信号通路奠定基础。     

L-Periaxin蛋白与Ezrin蛋白相互作用初步分析

Periaxin是施旺(Schwann)细胞中特异表达的支架蛋白之一,参与髓鞘成熟及稳定. Periaxin的基因缺失或突变导致脱髓鞘型腓骨肌萎缩症(CMT)4F亚型的发生. 埃兹蛋白(ezrin)是一种膜骨架连接蛋白,其在维持细胞形态和运动方面具有重要作用,与肿瘤细胞转移密切相关. 本文通过免疫共沉淀、双荧光共定位、YFP双分子荧光互补、GST-pull-down等实验方法,分析了L-periaxin蛋白与ezrin蛋白之间的相互作用. 结果表明,L-periaxin蛋白与ezrin蛋白可在细胞质中共定位并可发生相互作用.     

敲除L-periaxin基因的大鼠RSC96细胞系的建立

轴周蛋白(Periaxin)是外周神经系统非致密性髓鞘中特异表达的蛋白,编码Periaxin的基因经由选择性剪接产生两种蛋白异构体L-periaxin和S-periaxin,对髓鞘形成的初始化有重要作用。至今在Periaxin基因上已发现有18种不同的位点突变导致外周脱髓鞘神经疾病腓骨肌萎缩症4F亚型(CMT4F)的发生。利用转录激活因子样效应物核酸酶(TALENs)靶向基因敲除技术对大鼠RSC96细胞的periaxin基因进行敲除,根据TALENs设计原则,确定L-periaxin基因的敲除靶点在其编码NLS结构域部分,设计相应的TALEN左臂与右臂的识别序列,构建含上述识别序列的L-periaxin基因敲除载体TALEN-L和TALEN-R,并将其转入RSC96细胞,经嘌呤霉素药物筛选,获得L-periaxin基因敲除细胞株,经测序确认大鼠RSC96细胞中的基因组中L-periaxin基因区段已被敲除,成功构建了L-periaxin基因敲除细胞模型。计算L-periaxin基因敲除载体的突变率为21.6%。Western blotting实验证明,在RSC96细胞中只能检测到S-periaxin蛋白的表达。通过流式细胞术及MTT实验检测基因敲除细胞的细胞周期和生长速度,发现敲除L-periaxin基因的细胞生长速度缓慢,G1期细胞增多,S期细胞减少。     

L-periaxin与DRP2蛋白互作的精细分析

轴周蛋白L-periaxin作为外周神经系统中特异表达的骨架蛋白之一,在髓鞘成熟与维护过程中发挥重要作用.肌营养不良相关蛋白2(DRP2)是目前报道与L-periaxin存在明确相互作用的唯一蛋白,但两蛋白精细的互作区域及互作的分子机制仍不清楚.本研究通过双分子荧光互补、免疫共沉淀、GST pull-down、荧光光谱、细胞定位等技术,进一步揭示了periaxin蛋白的核定位信号NLS结构域上3段不同的亚结构域中,NLS1只参与periaxin蛋白的核定位,NLS2和NLS3参与DRP2蛋白的互作;DRP2蛋白中spectrin like2和WW结构域参与和L-periaxin蛋白的互作.L-periaxin与DRP2互作区域的精细定位,为进一步在蛋白水平探讨L-periaxin与脱髓鞘型腓骨肌萎缩症4F亚型的发生机制之间的关系提供参考.     

双分子荧光互补系统分析S-periaxin蛋白的聚合

S-periaxin蛋白是施旺氏细胞特异性表达的一种蛋白,在维持髓鞘的稳定方面发挥重要作用,该蛋白基因的突变引起腓骨肌萎缩症4F亚型的发生。Periaxin基因由于mRNA剪切方式的不同可以编码两种长短不同的含PDZ结构域的蛋白,即L-periaxin和S-periaxin。两种蛋白在施旺氏细胞的定位存在明显的差异,相对L-periaxin而言,S-periaxin无论是分子结构还是生物学功能均未见相关研究。该文从大鼠的施旺氏细胞系RSC96克隆了S-periaxin基因,构建了原核表达载体pETM-3C-S-periaxin,在大肠杆菌中进行重组表达,经Ni-NTA亲和柱和Sephacryl S-200凝胶层析柱获得电泳纯的目的蛋白。体外戊二醛交联分析蛋白的聚合状态表明,S-periaxin蛋白在体外易于形成不同聚合度的聚合物。免疫共沉淀也表明,S-periaxin蛋白存在同源蛋白间相互作用。另外,构建了原、真核双分子荧光互补系统,并利用该系统分析了细胞内S-periaxin蛋白间的相互作用。     

乏氧微环境、“瓦伯格”效应及上皮间质转化相关性

肿瘤细胞在氧气充足的情况下以糖酵解的方式供能,这一现象称为“瓦伯格”效应,被认为是肿瘤的第七大特征。上皮间质转化（epithelial mesenchymal transition,EMT）是一种重要的细胞过程,参与胚胎发育、伤口愈合及肿瘤的发生等过程中,被认为是恶性肿瘤的重要特征。近年研究表明,“瓦伯格”效应和上皮间质转化的发生均与肿瘤处于乏氧微环境密切相关。乏氧微环境除可直接诱导上皮间质转化发生外,还可诱导肿瘤细胞产生“瓦伯格”效应,进一步促进上皮间质转化的发生。本文就乏氧微环境、“瓦伯格”效应、以及上皮间质转化的相关性的研究进展做一综述,有助于揭示乏氧微环境、肿瘤能量代谢改变以及肿瘤迁移侵袭之间的因果关联。     

关于都市农业“菜篮子”工程发展建设的思考——以成都市为例

通过对成都市“菜篮子”产品与其他特4个大中心城市供给能力的对标分析、成都自身“菜篮子”市场的供需态势分析,探讨了具有典型都市现代农业特征的成都市“菜篮子”工程建设的基本现状与发展定位,为政府制定“菜篮子”工程发展战略提供参考依据。     

我国与“一带一路”沿线国家农产品贸易现状与合作前景

采用FAO数据库近年来农产品贸易数据,分析了“一带一路”沿线国家农产品贸易现状和中国与这些国家的贸易情况,并运用进出口依存指数对这些国家与我国贸易依赖程度进行了分析。在此基础上,就中国与“一带一路”沿线国家如何进一步开展贸易合作提出战略设想。     

基于文献分析视角的“一带一路”背景下农业发展研究热点分析

基于中国期刊全文数据库2013—2017年“一带一路”农业发展研究文献数据,利用内容分析和关键词词频分析方法,对“一带一路”倡议下农业发展领域的研究文献基础数据进行分析,对“一带一路”背景下农业领域当前研究热点进行分析,并在此基础上对今后的研究趋势进行预测。     

Ezrin interacts with L-periaxin by the “head to head and tail to tail” mode and influences the location of L-periaxin in Schwann cell RSC96

In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.     

Specific disruption of a schwann cell dystrophin-related protein complex in a demyelinating neuropathy.

Dystroglycan-dystrophin complexes are believed to have structural and signaling functions by linking extracellular matrix proteins to the cytoskeleton and cortical signaling molecules. Here we characterize a dystroglycan-dystrophin-related protein 2 (DRP2) complex at the surface of myelin-forming Schwann cells. The complex is clustered by the interaction of DRP2 with L-periaxin, a homodimeric PDZ domain-containing protein. In the absence of L-periaxin, DRP2 is mislocalized and depleted, although other dystrophin family proteins are unaffected. Disruption of the DRP2-dystroglycan complex is followed by hypermyelination and destabilization of the Schwann cell-axon unit in Prx(-/-) mice. Hence, the DRP2-dystroglycan complex likely has a distinct function in the terminal stages of PNS myelinogenesis, possibly in the regulation of myelin thickness.     

A tripartite nuclear localization signal in the PDZ-domain protein L-periaxin

The murine Periaxin gene encodes two PDZ-domain proteins in myelin-forming Schwann cells of the vertebrate peripheral nervous system (Dytrych, L., Sherman, D. L., Gillespie, C. S., and Brophy, P. J. (1998) J. Biol. Chem. 273, 5794-5800). Here we show that L-periaxin is targeted to the nucleus of embryonic Schwann cells. Subsequently, the protein redistributes to the plasma membrane processes of the myelinating Schwann cell where it is believed to function in a signaling complex. In contrast, L-periaxin remains in the nucleus when expressed ectopically in oligodendrocytes, the myelin-forming glia of the central nervous system. The nuclear localization signal (NLS) is basic and tripartite and comprises three signals that act synergistically. Nuclear targeting of L-periaxin is energy-dependent and is inhibited by cell-cell contact. These data show that L-periaxin is a member of a growing family of proteins that can shuttle between the nucleus and cortical signaling/adherence complexes.     

Cellular contacts in myelinated fibers of the peripheral nervous system

Myelination allows the fast propagation of action potentials at a low energetic cost. It provides an insulating myelin sheath regularly interrupted at nodes of Ranvier where voltage-gated Na+ channels are concentrated. In the peripheral nervous system, the normal function of myelinated fibers requires the formation of highly differentiated and organized contacts between the myelinating Schwann cells, the axons and the extracellular matrix. Some of the major molecular complexes that underlie these contacts have been identified. Compact myelin which forms the bulk of the myelin sheath results from the fusion of the Schwann cell membranes through the proteins P0, PMP22 and MBP. The basal lamina of myelinating Schwann cells contains laminin-2 which associates with the glial complex dystroglycan/DPR2/L-periaxin. Non compact myelin, found in paranodal loops, periaxonal and abaxonal regions, and Schmidt-Lanterman incisures, presents reflexive adherens junctions, tight junctions and gap junctions, which contain cadherins, claudins and connexins, respectively. Axo-glial contacts determine the formation of distinct domains on the axon, the node, the paranode, and the juxtaparanode. At the paranodes, the glial membrane is tightly attached to the axolemma by septate-like junctions. Paranodal and juxtaparanodal axoglial complexes comprise an axonal transmembrane protein of the NCP family associated in cis and in trans with cell adhesion molecules of the immunoglobulin superfamily (IgSF-CAM). At nodes, axonal complexes are composed of Na+ channels and IgSF-CAMs. Schwann cell microvilli, which loosely cover the node, contain ERM proteins and the proteoglycans syndecan-3 and -4. The fundamental role of the cellular contacts in the normal function of myelinated fibers has been supported by rodent models and the detection of genetic alterations in patients with peripheral demyelinating neuropathies such as Charcot-Marie-Tooth diseases. Understanding more precisely their molecular basis now appears essential as a requisite step to further examine their involvement in the pathogenesis of peripheral neuropathies in general.     

Correction

The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt–Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32-null mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.     

Emerging role for ERM proteins in cell adhesion and migration

The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.Key words: epithelial cells, membrane-cytoskeleton interface, morphogenesis, ERM proteins, cell adhesion