Complexin促进神经母瘤细胞分化的作用机制

Complexin(Cpx)蛋白是一种参与神经元囊泡释放过程的小分子蛋白,CpxⅠ对于神经细胞分化的促进作用主要依赖于其促进囊泡同步释放的功能,但其他亚型的Cpx及不同物种中的Cpx同源类似物是否也具有促进神经细胞分化的功能尚未被阐明。在本研究中,我们在小鼠的神经母瘤细胞(N2a)中过表达不同亚型和种属的Cpx蛋白,24 h和48 h后分别统计分化率、分支数和分支长3个指标。结果表明,不同亚型和不同种属物种的Cpx对神经母瘤细胞的分化均具有促进作用,这说明Cpx通过促进囊泡同步释放功能促进神经细胞分化的作用是保守的。     

Syntaxin蛋白对神经母瘤细胞分化的影响及机制研究

Syntaxin是特异性地分布在神经细胞突触前质膜上的一种多结构域蛋白,它是细胞质膜融合的关键性蛋白,但Syntaxin在神经细胞分化过程中的作用尚未阐明。本实验旨在探讨Syntaxin蛋白对神经母瘤细胞分化的影响及其影响机制。通过在小鼠的神经母瘤细胞（N2a）中过表达不同的Syntaxin蛋白突变体,统计细胞的分化率、突起分支数和突起总长度等参数,来观察Syntaxin蛋白及其突变体对神经细胞分化的影响。通过实验结果得知Syntaxin蛋白促进神经母瘤细胞分化的作用位点在其氨基端的Habc结构域,主要影响细胞的分化突起数目和突起总长度,对细胞分化率无显著作用。     

抗病毒固有免疫分子TRIM22 C端SPRY结构域对其转录、翻译及亚细胞定位的影响

本文旨在探讨TRIM22 C端SRPY结构域对其转录、翻译及亚细胞定位的影响。以野生型TRIM22真核表达质粒为模板,扩增出C端SPRY结构域缺失的TRIM22基因片段,并将其克隆入真核表达质粒pcDNA3.1。将野生型和突变型TRIM22真核表达载体分别转染高分化人肝癌细胞株HepG2。通过反转录-聚合酶链反应检测其mRNA表达水平,蛋白质免疫印迹法(Western blot)检测其蛋白表达水平,并通过间接免疫荧光法检测其亚细胞内定位。结果成功构建了C端SPRY结构域缺失的TRIM22真核表达质粒。转染HepG2细胞后,TRIM22 SPRY结构域缺失突变体在转录和翻译水平上均与野生型TRIM22无明显差异,但TRIM22 SPRY结构域缺失突变体完全丧失了核定位能力,这为进一步研究SPRY结构域在TRIM22抗病毒过程中所发挥的作用及机制提供了有益的启示。     

具有八肽重复区突变的朊蛋白其抗氧化性降低

抗氧化性被认为是细胞朊蛋白的主要生理功能之一,研究显示它的抗氧化性主要与朊蛋白序列中的八肽重复区有关．但是迄今为止它的抗氧化机制仍旧不清楚．我们构建表达了野生型朊蛋白（PrP-PG5）和它的不同八肽重复区突变体0（PrP-PG0）,9（PrP-PG9）和12（PrP-PGl2）．各种原核表达突变体蛋白在H202氧化后出现分子量的增加,并可导致羰基产生．MTT和细胞计数实验显示表达各种突变体的细胞存活率明显低于表达野生型朊蛋白（PrP—PG5）的细胞．细胞内ROS检测发现表达各种突变体的细胞内ROS水平明显高于表达野生型朊蛋白（PrP-PG5）的细胞．此外,谷胱甘肽过氧化物酶检测显示表达野生型朊蛋白（PrP-PG5）的细胞内谷胱甘肽过氧化物酶水平明显高于表达各种突变体的细胞．H2O2处理细胞后,转染突变体的细胞总的羰基产物数量明显高于转染野生型朊蛋白（PrP-PG5）的细胞,而表达突变体细胞及转染空载体的细胞较表达野生型朊蛋白（PrP-PG5）的细胞对氧化物质的抵抗性明显减弱．这些结果提示,具有正确八肽重复区数目对于朊蛋白（PrP）的抗氧化作用起关键作用,PrP的抗氧功能的丢失可能参与家族性朊病毒病的病理过程．     

GDNF缺失突变体的设计及其结构与功能关系(英文)

研究了GDNF结构与功能的关系 .基于鼠源GDNF的晶体结构 ,利用计算机SGIIndigo2(R4 4 0 0 )工作站和InsightⅡ (95.5)蛋白质分析软件模拟了人GDNF三维结构 ,设计了GDNF分子的两个缺失突变体ΔN1 2 8和ΔN78 90 .以野生型GDNFcDNA作为模板 ,用PCR法得到编码缺失突变体的DNA片段 .将大肠杆菌作为表达系统 ,使缺失突变体GDNF在大肠杆菌中表达 ,对表达产物纯化和复性后进行生物活性测定 .两株突变体在大肠肝菌中获得了高效表达 ,纯化后的GDNF突变体ΔN1 2 8可以与存在于KG 1a细胞表面的受体结合 ,但不能促进 8日龄鸡胚背根节突起的生长 .突变体ΔN78 90既不能与受体结合 ,同时也失去了促背根节突起生长的功能 .说明GDNF分子的N端氨基酸对分子的生物学活性很重要 ,但对分子与受体GDNFR α的结合并不是必需的 ,而分子中的螺旋区对分子与受体的结合以及生物学活性都必不可少 .     

Sim2基因对PC12细胞分化的影响

[摘要] 目的 探讨位于Down综合征关键位点的Sim2基因对PC12细胞分化的影响及其机制。 方法 以pcDNA3-mSim2真核表达载体稳定转染PC12细胞,以倒置相差显微镜镜观察PC12细胞神经突起的变化;以RT-PCR方法检测神经元分化相关基因GAP43和Synapsin I mRNA表达水平的变化;流式细胞仪检测GAP43蛋白的表达。 结果 RT-PCR结果显示, pcDNA3-mSim2转染后,mSim2 mRNA表达明显上调;与对照组相比,转染mSim2的PC12细胞突起数量显著减少,长度明显变短;GAP43和Synapsin I mRNA表达水平明显降低（P<0.05）;流式细胞仪检测发现,转染mSim2的PC12细胞GAP43蛋白表达水平显著降低（P<0.05）。 结论 Sim2基因可通过影响神经元的分化参与Down综合征的发生。     

酿酒酵母GPCR蛋白Ste2亚细胞定位信号探索

G蛋白偶联受体(G protein-coupled receptor,GPCR)家族蛋白在细胞感受各种胞外信号过程中发挥重要作用。Ste2是酵母细胞中GPCR蛋白之一。大量文献报道了Ste2蛋白突变体对其功能和表达的影响,但关于Ste2亚细胞定位的研究相对较少。这项工作的目的在于确定Ste2亚细胞定位,探究Ste2不同跨膜域、胞内外环状结构域和N端、C端对其亚细胞定位的影响。构建了一系列结构域删除或替换突变体,通过荧光显微镜观察判断不同结构区域对Ste2亚细胞定位的影响,并通过与已知的细胞器标记蛋白共定位观察验证亚细胞定位判读结果。结果显示:野生型Ste2荧光信号出现在质膜和液泡内腔; C端缺失突变体荧光信号出现在质膜和内质网。在N端、C端、各环状结构域序列采用动物GPCR蛋白ORI7、OR17-40相应结构域替换的突变体中,C端替换导致液泡内腔信号消失,质膜信号强于野生型; N端和部分环状结构域替换不同程度减弱或消除了质膜定位,液泡腔内信号类似于野生型;部分突变体在胞内出现点状分布的荧光信号。由此推断:Ste2 N端,第一、第二胞外环状结构域和第三胞内环状结构域可能具有影响Ste2运输定位到质膜的功能;而C端则可能在Ste2离开细胞膜进入液泡的过程中发挥作用。初步确定了Ste2的不同结构区域对其定位的影响,为深入研究GPCR蛋白的亚细胞定位机制奠定基础。     

人SDCT2蛋白亚细胞定位信号分析

为了确定人高亲和力钠离子依赖性二羧酸共转运蛋白（high-affinity sodium-dependent dicarboxylate co-transporter, SDCT2,NaDC3）在细胞内的定位,构建了SDCT2与增强型绿色荧光蛋白（EGFP）的融合蛋白表达载体,并转染肾小管上皮细胞LLC-PK1,激光共聚焦显微镜观察显示,SDCT2蛋白主要定位于细胞的基底侧膜上.同时将SDCT2-EGFP融合基因mRNA显微注射到爪蟾卵母细胞中表达,可见融合蛋白的绿色荧光仅分布在细胞膜上.为了进一步确定该蛋白质的亚细胞定位信号序列,将SDCT2基因的N端及C端分别缺失,并构建缺失突变体与EGFP的融合蛋白表达载体,将它们转染到LLC-PK1中,观察SDCT2 缺失体在细胞内的分布情况.结果显示,N端缺失的SDCT2蛋白主要位于细胞质中,顶膜和基底侧膜上也有表达;C端缺失的SDCT2蛋白主要位于基底侧膜上,顶膜几乎没有表达,细胞质中表达很少.免疫组化结果也显示,SDCT2只表达于人近端肾小管上皮细胞的基底侧膜.这表明SDCT2蛋白的N端序列对其亚细胞定位是必需的,人SDCT2蛋白的基底膜定位信号位于N端序列中.     

neuronatin shRNA真核表达载体的构建及其生物学功能

目的:获得能持续干扰neuronatin（nnat）基因表达的细胞,观察nnat基因沉默对神经细胞发育与分化的影响,为研究基因功能奠定基础。方法:构建含nnat基因短发夹RNA(shRNA)表达质粒,将质粒转染大鼠肾上腺嗜铬细胞瘤细胞PC12,RT-PCR方法筛选出最有效干扰质粒,稳定转染PC12细胞后观察细胞表型变化,免疫荧光检测nnat蛋白表达,NGF诱导观察nnat表达下调对细胞分化的影响。结果:成功构建并筛选出有效的靶向nnat基因的shRNA真核表达载体;载体稳定转染PC12细胞之后能特异性沉默nnat基因的表达,PC12细胞长出突起,向神经元方向分化,加入诱导因子NGF后能促进突起生长。结论:nnat可能是作为神经分化抑制因子在神经发育与成熟过程中发挥作用。     

敲除Pofut1不影响胚胎干细胞向神经元分化

蛋白O-连接岩藻糖基转移酶1 (Pofut1)基因缺失可导致Notch分子无法与配体结合并启动信号传递. 为研究Pofut1基因对哺乳动物胚胎干细胞（ESC）向神经分化的影响,利用Pofut1基因敲除的胚胎干细胞与野生型胚胎干细胞,经体外培养诱导拟胚体（EB）分化为神经细胞,计数分化为神经细胞的比例,采用细胞免疫组化染色和real-time PCR等方法,分析神经细胞特异性标志分子的表达. 结果显示,Pofut1基因缺失后,对EBC生长没有明显影响,分化过程中形成的拟胚体数量明显增多,分化的神经样细胞以及神经标志物分子的表达也明显多于对照组;Notch信号缺失对小鼠胚胎干细胞生长无明显影响,但可以促进ES细胞向神经细胞分化.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

The Cpx two-component signal transduction pathway is activated in Escherichia coli mutant strains lacking phosphatidylethanolamine.

The CpxA-CpxR two-component signal transduction pathway of Escherichia coli was studied in a mutant (pss-93) lacking phosphatidylethanolamine (PE). Several properties of this mutant are comparable to phenotypes of cpxA point mutants, indicating that this two-component pathway is activated in PE-deficient cells. In contrast to point mutants, cpx operon null mutants have a wild-type phenotype. By use of this information, a cpx operon null allele was introduced into a pss-93 mutant. Certain altered properties of PE-deficient mutants, which were consistent with activation of the Cpx pathway, returned to the wild-type phenotype, namely, active accumulation of proline and thiomethyl-beta-D-galactopyranoside was partially restored to wild-type levels, increased resistance to amikacin returned to wild-type sensitivity, and high levels of degP expression returned to repressed wild-type levels. Elevated levels of acetyl phosphate and nlpE gene product can result in activation of the Cpx pathway. However, inactivation of the nlpE gene or mutations eliminating the ability to make acetyl phosphate did not alter the high level of degP expression in pss-93 mutants. We propose that the lack of PE results in an alteration in cell envelope structure or physical properties, leading to direct activation of the Cpx pathway.     

N-terminal deletion does not affect α-synuclein membrane binding, self-association and toxicity in human neuroblastoma cells, unlike yeast

α-Synuclein causes Parkinson's disease if mutated or aberrantly produced in neurons. α-Synuclein-lipid interactions are important for the normal function of the protein, but can also contribute to pathogenesis. We previously reported that deletion of the first 10 N-terminal amino acids dramatically reduced lipid binding in vitro, as well as membrane binding and toxicity in yeast. Here we extend this study to human neuroblastoma SHSY-5Y cells, and find that in these cells the first 10 N-terminal residues do not affect α-synuclein membrane binding, self-association and cell viability, contrary to yeast. Differences in lipid composition, membrane fluidity and cytosolic factors between yeast and neuronal cells may account for the distinct binding behavior of the truncated variant in these two systems. Retinoic acid promotes differentiation and α-synuclein oligomer formation in neuroblastoma cells, while addition of a proteasomal inhibitor induces neurite outgrowth and toxicity to certain wild-type and truncated α-synuclein clones. Yeast recapitulate several features of α-synuclein (patho)biology, but its simplicity sets limitations; verification of yeast results in more relevant model systems is, therefore, essential.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.