人微丝相关蛋白hHBRK1突变体的亚细胞定位

 hHBrk1是本研究室利用抑制性消减杂交手段,从人支气管上皮细胞恶性转化株BERP3 5中克隆到的差异高表达基因.hHBRK1蛋白家族序列在动、植物界高度保守,含有一个7重复 (heptad repeat, HR) 结构域.利用绿色荧光蛋白(GFP)报告系统,发现野生型hHBRK1蛋白在胞浆中弥散分布,在细胞运动前沿富集,与细胞片状伪足的微丝共定位.hHBRK1- R54和hHBRK1-S56 G57蛋白在胞浆弥散分布,但失去了在细胞运动前沿富集的特征.hHBRK1ΔN(1-45)在细胞内弥散分布,而hHBRK1ΔC(46-75)选择性地在高尔基体富集.研究提示,hHBRK1蛋白为微丝相关蛋白,结构的完整性是其发挥功能的前提.hHBRK1蛋白可能通过HR结构域调控微丝聚合,从而参与微丝依赖性的细胞运动或物质运输.     

微丝相关蛋白HSPC300/hHBRK1与肌球蛋白Ⅵ相互作用的鉴定

为鉴定微丝相关蛋白HSPC300/hHBRK1在肝脏组织的功能,采用GST pull-down结合质 谱技术,检测该蛋白在肝脏中的结合蛋白,结果提示,肌球蛋白Ⅵ与HSPC300/hHBRK1共沉降,Western 印迹杂交证实了质谱的结果．构建HSPC300/hHBRK1原核表达载体,诱导并获得了His-hHBRK1融合蛋白．利用免疫共沉淀证实hHBRK1与肌球蛋白Ⅵ存在相互作用,激光共聚焦检测显示hHBRK1与肌球蛋白Ⅵ在肺癌95D细胞的胞浆共定位,提示其相互作用可能是直接结合．肌球蛋白Ⅵ参与细胞迁移、高尔基分泌泡的运输和维持高尔基体稳定性等作用．hHBRK1与肌球蛋白Ⅵ相互作用,为微丝相关蛋白HSPC300/hHBRK1参与细胞迁移和胞内物质运输提供了进一步佐证．     

皮层蛋白介导斑马鱼(Danio rerio)原肠化细胞运动

在胚胎发育过程中, 细胞运动对指导原肠期胚胎细胞的时空定位并决定其发育命运具有核心作用, 然而活体状态下原肠化过程中细胞运动的调控机制目前并不清楚. 微丝结合蛋白皮层蛋白(cortactin)是微丝核化过程的重要调控分子, 它通过激活微丝相关蛋白2/3复合物(Arp2/3 complex)促进微丝在细胞前导缘区域迅速组装, 从而直接作用于细胞运动. 为阐明斑马鱼(Danio rerio)原肠化细胞运动的分子调控机制, 本研究首先检测了皮层蛋白在斑马鱼胚胎发育过程的表达水平. Western blotting分析证明皮层蛋白在斑马鱼原肠期胚胎中大量表达; 整装胚胎抗体染色结果表明在斑马鱼原肠化过程中, 皮层蛋白主要分布于胚胎背侧胚盾区域的细胞中, 在发生活跃运动的上皮层细胞和下皮层细胞中含量较高;在亚细胞水平, 皮层蛋白和Arp2/3复合物共同定位于运动的皮层区域, 并在细胞连接处也有大量分布. 此外, 研究还发现皮层蛋白在发育中的中枢神经系统中表达量较高. 本研究结果首次表明皮层蛋白和Arp2/3复合物介导的微丝聚合参予了斑马鱼原肠化细胞运动, 并在中枢神经系统发育中扮演重要角色.     

脑红蛋白和细胞红蛋白：携氧蛋白质家族2个新成员

脑红蛋白(neuroglobin, Ngb)和细胞红蛋白(cytoglobin, Cygb)是新发现的2个携氧蛋白家族的成员.脑红蛋白主要存在于脑中,而细胞红蛋白在全身各个组织都含有,它们和另外2个携氧蛋白——血红蛋白和肌红蛋白的同源性<25%,但它们在种属之间的同源性很高(>95%).脊椎动物脑红蛋白基因定位于14q24,细胞红蛋白基因定位于17q25,都含有4个外显子和3个内含子.2种蛋白在生理条件下含有6个配位键,不同于血红蛋白和肌红蛋白的5个配位键结构.这2种新蛋白和氧都具有很高的亲和力,在缺氧条件下其基因及蛋白表达都有明显的提升,对细胞的存活有一定保护作用.对于脑红蛋白和细胞红蛋白的功能研究,有助于更好地了解机体氧代谢和氧利用过程,并为临床在缺氧损伤时的治疗提供新的观点和途径.     

人CREB1基因及蛋白的生物信息学分析

[目的]利用生物信息学方法预测分析人CREB1基因的启动子情况及蛋白质的理化性质、亲疏水性、细胞亚定位、蛋白质多级结构、与之相互作用的蛋白质以及GO注释。[方法]利用相应软件及网站预测分析CREB1基因及其蛋白的相关信息。[结果]发现CREB1基因存在4个启动子,其转录受SP1和AP-2影响较大;其蛋白质是由431个氨基酸组成的主要定位于细胞核的不稳定亲水蛋白,其等电点为5.44,序列在多个物种间保守性强;二级结构主要由α螺旋和无规卷曲;需要更多可靠的模板构建三级结构;CREB1调控由c AMP介导的信号通路,对神经系统疾病及肿瘤的发生发展有重要作用。[结论]SP1和AP2通过调控多个启动子影响CREB1的表达,其蛋白质主要定位于细胞核,通过调控c AMP介导的信号通路维持人体正常生理状态。     

酿酒酵母中钙信号途径对crz1调控基因表达的影响

该文通过对酿酒酵母细胞中120个钙离子敏感性基因的启动子进行序列分析发现,24个基因的启动子上含有转录因子Crz1的结合位点(GNG GC[T/G]CA或GNG GCT G)。该研究检测了钙信号途径对24个基因表达和细胞定位的影响。实验结果表明,钙信号途径通过转录因子Crz1诱导19个基因的特异性表达及其编码蛋白的细胞定位。结果发现,5个基因的功能与代谢相关(TPS1、PHO86、ERG3、ARG82和AKR1)、5个基因的功能与离子稳态相关(CSG2、PMC1、VMA10、MNR2和VAM2)、4个基因的功能与蛋白质分选相关(PEP3、VPS36、VPS27和VPS4)以及5个基因的功能与转录相关(DEP1、IMP2′、THP1、SGF29和ROX3)。该文的研究结果为研究酿酒酵母细胞中钙离子稳态调控机制提供了理论基础。     

苦荞转录因子FtMYBF的克隆、亚细胞定位及表达分析

R2R3-MYB转录因子SG7亚家族成员在植物黄酮醇生物合成中具有极其重要的调控作用。探究苦荞中SG7 R2R3-MYB转录因子在黄酮醇生物合成中的功能,为苦荞黄酮醇生物合成的分子调控机制研究奠定基础。采用RT-PCR技术从苦荞中克隆到一个前期经转录组与代谢组联合分析鉴定到的SG7 R2R3-MYB转录因子,命名为FtMYBF。利用生物信息学、亚细胞定位、基因共表达、基因表达量与总黄酮含量相关性对该基因进行了分析。结果表明,FtMYBF全长CDS序列为1119 bp,编码372个氨基酸。蛋白多序列比对和系统进化树分析表明,FtMYBF属于R2R3-MYB SG7亚家族成员。亚细胞定位显示,FtMYBF蛋白定位于细胞核。基因共表达分析表明,FtMYBF与多个苦荞黄酮醇生物合成结构基因共表达。基因表达量与总黄酮含量间相关性分析表明,FtMYBF在不同组织部位表达量与总黄酮含量高度正相关。FtMYBF属于R2R3-MYB SG7亚家族成员,是一个核定位转录因子,其可能通过正调控苦荞黄酮醇生物合成结构基因的表达来正调控苦荞黄酮和黄酮醇的生物合成。     

小细胞肺癌中转录因子E2F1调控的靶基因（英文）北大核心CSCD

本研究组前期研究结果表明,转录因子E2F1在大约95%的小细胞肺癌组织中表达上调,而且与其浸润、转移密切相关,但是E2F1在小细胞肺癌中调控的靶基因未见报道。本研究旨在探索E2F1在小细胞肺癌细胞株H1688中调控的靶基因。染色体免疫共沉淀联合测序(chromatin immunoprecipitation sequencing,Ch IP-seq)结果显示,在小细胞肺癌H1688细胞中,E2F1能够调控5 326个靶基因的表达,其中4 700个是结构基因,626个基因编码长链非编码RNA。基因功能注释(gene ontology,GO)和基因富集图谱(enrichment map)分析显示,E2F1调控的靶基因功能主要集中在3个方面:细胞周期调控、染色体和组蛋白修饰以及蛋白转运。MEME4.7.0软件分析显示,E2F1通过结合6个序列调控相关靶基因和长链非编码序列的表达。以上结果阐明了E2F1在小细胞肺癌中调控的靶基因,为进一步研究E2F1在小细胞肺癌发生、发展、浸润与转移、复发和耐药中的作用提供了实验依据。     

‘741杨’D2类细胞周期蛋白基因的克隆及功能鉴定

D类细胞周期蛋白(D-type cyclin,CYCD)调控细胞周期G1/S期转变。CYCD与细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK)结合形成CYCD/CDK复合物,被激活的CYCD/CDK复合物通过磷酸化下游细胞周期响应因子调控细胞周期有序进行,进而影响植物的生长发育。该研究以‘741杨’为实验材料,成功鉴定得到1个D2类细胞周期蛋白基因(PtoCYCD2;1)。研究表明:(1)实时定量PCR(qRT-PCR)显示,PtoCYCD2;1基因在根、茎、叶、叶柄、树皮和木质部中均有表达,在叶中的相对表达水平最高。(2)亚细胞定位表明PtoCYCD2;1蛋白定位于细胞核。(3)与野生型(Wild-Type poplar,WT)相比,过表达PtoCYCD2;1基因的‘741杨’出现株高降低,茎直径减小,叶片明显下卷的表型。(4)扫描电镜分析(SEM)显示,转基因杨树叶片的上表皮细胞平均面积变小,细胞数量增多;树脂切片结果显示与WT相比,转基因杨树叶片的栅栏组织和海绵组织的细胞间隙疏松。(5)qRT-PCR结果显示,转基因杨树中细胞周期调控基因CDKA;1、CDKB1;1和CDKB2;1的表达水平显著上调,植物成视网膜细胞瘤相关蛋白1(retinoblastoma-related protein1,RBR1)基因、细胞周期蛋白依赖性激酶抑制因子(kip-related protein,KRP)基因的表达水平显著下调。该研究结果为进一步研究木本植物CYCD2基因的功能奠定了基础。     

小细胞肺癌中转录因子E2F1调控的靶基因（英文）

本研究组前期研究结果表明,转录因子E2F1在大约95%的小细胞肺癌组织中表达上调,而且与其浸润、转移密切相关,但是E2F1在小细胞肺癌中调控的靶基因未见报道。本研究旨在探索E2F1在小细胞肺癌细胞株H1688中调控的靶基因。染色体免疫共沉淀联合测序(chromatin immunoprecipitation sequencing,Ch IP-seq)结果显示,在小细胞肺癌H1688细胞中,E2F1能够调控5 326个靶基因的表达,其中4 700个是结构基因,626个基因编码长链非编码RNA。基因功能注释(gene ontology,GO)和基因富集图谱(enrichment map)分析显示,E2F1调控的靶基因功能主要集中在3个方面:细胞周期调控、染色体和组蛋白修饰以及蛋白转运。MEME4.7.0软件分析显示,E2F1通过结合6个序列调控相关靶基因和长链非编码序列的表达。以上结果阐明了E2F1在小细胞肺癌中调控的靶基因,为进一步研究E2F1在小细胞肺癌发生、发展、浸润与转移、复发和耐药中的作用提供了实验依据。     

微丝相关新蛋白hHBRK1相互作用蛋白质的鉴定

为了鉴定hHBRK1的相互作用蛋白,通过DNA重组构建重组表达质粒pGEXhHBRK1,并以谷胱甘肽Sepharose4B亲合层析法,获得纯化的重组融合蛋白GSThHBRK1.以小鼠心肌组织为研究对象,采用GSTpulldown技术结合Western印迹法,证实hHBRK1与小鼠心肌肌钙蛋白TEa亚型(EacTnT)相互作用.结果提示,hHBRK1与EacTnT结合,可能参与心肌微丝的聚合,为小鼠cTnT众多的剪接体,提供了一种可能的功能定位.     

Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.     

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.     

Role of actin cytoskeletal dynamics in activation of the cyclic AMP pathway and HWP1 gene expression in Candida albicans

Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.     

F-actin慢生长端的盖帽蛋白:红细胞原肌球调节蛋白

红细胞原肌球调节蛋白(erythrocyte tropomodulin,E-Tmod)是从红细胞膜中提取的原肌球蛋白(tropomyosin,TM)的结合蛋白.其N-端有两个TM结合位点和一个TM依赖的actin结合位点,C-端有5个富含亮氨酸的重复序列和一个TM非依赖的actin结合位点.作为F-actin慢生长端唯一的盖帽蛋白,E-Tmod与TM的N-端结合并同时与actin结合,减慢由TM包被的F-actin的解聚速度.E-Tmod编码基因高度保守,在红细胞、心肌细胞等细胞中广泛表达.E-Tmod对于F-actin和细胞骨架的组织以及对细胞力学特性的保持具有至关重要的作用.     

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.     

Identification of phostensin, a PP1 F-actin cytoskeleton targeting subunit

We have identified a novel protein, protein phosphatase 1 F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase 1 (PP1) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PP1 complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin-Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PP1 to F-actin cytoskeleton. The phostensin/PP1 complex may play a vital role in modulation of actin rearrangements.     

Neurabin II mediates doublecortin-dephosphorylation on actin filaments

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder. DCX binds to microtubules and actin filaments. Association of Dcx with F-actin is regulated by site-specific phosphorylation and by neurabin II, an F-actin binding protein that also binds to Dcx. We show here that neurabin II mediates dephosphorylation of Dcx by protein phosphatase 1 (PP1). Furthermore, overexpression of PP1 reduces Dcx phosphorylation and decreases Dcx binding to F-actin. By contrast, abolishing PP1 binding to neurabin II maintains phosphorylation levels of Dcx, leading to a retention of Dcx at F-actin. We suggest that a dynamic regulation of Dcx mediated by neurabin II regulates the translocation of Dcx from F-actin to microtubules and vice versa.     

Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.