Rac GTPase-activating protein (Rac GAP) α1-Chimaerin undergoes proteasomal degradation and is stabilized by diacylglycerol signaling in neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.     

Rac1 modulation of the apical domain is negatively regulated by β (Heavy)-spectrin

Epithelial polarity and morphogenesis require the careful coordination of signaling and cytoskeletal elements. In this paper, we describe multiple genetic interactions between the apical cytoskeletal protein β(H) and Rac1 signaling in Drosophila: activation of Rac1 signaling by expression of the exchange factor Trio, is strongly enhanced by reducing β(H) levels, and such reductions in β(H) levels alone are shown to cause an increase in GTP-Rac1 levels. In contrast, co-expression of a C-terminal fragment of β(H) (βH33) suppresses the Trio expression phenotype. In addition, sustained expression of βH33 alone in the eye induces a strong dominant phenotype that is similar to the expression of dominant negative Rac1(N17), and this phenotype is also suppressed by the co-expression of Trio or by knockdown of RacGAP50C. We further demonstrate that a loss-of-function allele in pak, a Rac1 effector and negative regulator of β(H)' dominantly suppresses larval lethality arising loss-of-function karst (β(H)) alleles. Furthermore, expression of constitutively active Pak(myr) in the larval salivary gland induces expansion of the apical membrane and destabilization of the apical polarity determinants Crumbs and aPKC. These effects resemble a Rac1 activation phenotype and are suppressed by βH33. Together, our data suggest that apical proteins including β(H) are negatively regulated by Rac1 activation, but that Rac1 signaling is also suppressed by β(H) through its C-terminal domain. Such a system would be bistable with either Rac1 or β(H) predominant. We suggest a model for apical domain maintenance wherein Rac1 down-regulation of β(H) (via Pak) is opposed by β(H)-mediated down-regulation of Rac1 signaling.     

Rac1、Rac2参与调节人单核细胞趋化和NADPH氧化酶活性

为了探讨小分子GTP酶蛋白Rac1和Rac2在人单核细胞中趋化迁移以及还原型辅酶II（NADPH）氧化酶活性中的作用,采用小分子干扰siRNA对人单核细胞中RAC1、RAC2分别进行特异性抑制,采用实时定量PCR技术、免疫印迹技术在RNA和蛋白质水平上确认抑制效果,使用甲酰三肽(formyl-met-leu-phe,fMLP)、人单核细胞趋化因子（monocyte chemoattractant protein-1,MCP-1）诱导单核细胞趋化;用血清调理的酵母多糖（serum opsonized zymosan,ZOP)、佛波酯（phosphomolybdic acid, PMA）激活单核细胞NADPH氧化酶活性,诱导活性氧(Reactive oxygen species, ROS)产生,以此对Rac1和Rac2作用进行研究. 结果表明,小分子干扰siRNA能够在mRNA水平和蛋白质水平分别有效抑制目的基因表达;使用Chamber assay方法发现,仅Rac1参与了fMLP、MCP-1诱导的人单核细胞趋化. Rac激活实验确证,Rac1参与MCP-1诱导的趋化;细胞色素C还原法表明,Rac1和Rac2均参与PMA和ZOP诱导人单核细胞ROS生成. 在人单核细胞中,RAC1和RAC2基因沉默模型的成功建立以及初步研究显示,Rac1和Rac2的不同作用结果将为深入研究它们在人单核细胞中的功能奠定了良好基础.     

Rac activation: P-Rex1 - a convergence point for PIP(3) and Gbetagamma?

P-Rex1, a novel Rac activator, has been identified in the first biochemical purification of a guanine nucleotide exchange factor for GTPases of the Rho family. P-Rex1 is synergistically activated by PIP(3) and Gbetagamma and may act as a coincidence detector for these signaling molecules.     

Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas^V12) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas^L61, but not KRas^V12, also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas^V12 and KRas^L61 gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.     

Structure of Poly (dT)·Poly (dA)·Poly (dT)

Abstract

The molecular structure of poly (dT)·poly (dA)·poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right- handed triple-helix of pitch 38.4 Å and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2′-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

Rac的研究进展

Ｒｈｏ家族小Ｇ蛋白 (Ｒｈｏ ＧＴＰａｓｅ)参与细胞骨架重组、基因转录调控和细胞信号转导等生理过程 ,从而影响细胞的极性、粘附、生长和运动能力。有证据表明Ｒｈｏ家族成员还参与肿瘤细胞的侵袭和转移。Ｒａｃ是Ｒｈｏ家族小Ｇ蛋白的成员之一 ,有Ｒａｃ1、Ｒａｃ2、Ｒａｃ3 3种同工型形式。Ｒａｃ1和Ｒａｃ3在体内分布广泛 ,而Ｒａｃ2是一种造血细胞特异的小Ｇ蛋白 ,它可通过对ＮＡＤＰＨ氧化酶的作用调节超氧化物的产生。Ｒａｃ有二种存在形式 :有活性的ＧＴＰ结合形式与无活性的ＧＤＰ结合形式。Ｒａｃ和其他小Ｇ蛋白一样 ,可被鸟嘌呤…     

Structures for the polynucleotide complexes poly(dA) · poly(dT) and poly(dT) · poly(dA) · poly(dT)

X-ray diffraction analyses of fibers of polydeoxyadenylic acid · polydeoxythymidylic acid show that this molecule exists as a 10-fold double-helix with axial rise per nucleotide $\text{h = 3.24}\text{to}\text{3.29}\text{A}\text{?}$. The structure is very similar to B-DNA ($\text{h = 3.37}\text{A}\text{?}$) in having C3-exo furanose rings and base-pairs positioned centrally on the helix axis, but distinctive enough to have two packing modes, neither of which has been observed for B-DNA. Although the triple-stranded poly(dT) · poly(dA) · poly(dT) also has a large value of h(3.26 Å), each of the chains is a 12-fold helix of the A-genus with C3-endo furanose rings and bases displaced several Angstrom units from the helix axis.     

Differential expression of ·1(IV), ·2(IV), ·5(IV) and ·6(IV) collagen chains in the basement membrane of basal cell carcinoma

Type IV collagen, the major component of basement membrane, consists primarily of ·1(IV) and ·2(IV) chains. Recently, other types of collagen IV chains, i.e. ·3(IV), ·4(IV), ·5(IV) and ·6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of ·(IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six ·(IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, ·1(IV) and ·2(IV) chains were discontinuously stained, and ·5(IV) and ·6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, ·1(IV), ·2(IV), ·5(IV) and ·6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, ·1(IV) and ·2(IV) chains were continuously stained, and ·5(IV) and ·6(IV) chains were discontinuous or absent. The assembly of ·5(IV) and ·6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma This revised version was published online in November 2006 with corrections to the Cover Date.     

抑制Rac1蛋白活化阻碍胚胎发育早期血管新生

小G蛋白Rac1在胚胎发育早期血管形成尤其是内皮发生过程中的作用尚不清楚．采用胚胎干细胞(ESCs)为模型,建立稳定表达持续表达型Rac1(G12V)和显性失活型Rac1(T17N)编码序列的小鼠ESCs并制备胚胎小体(EBs),诱导分化后观察Rac1(G12V)和Rac1(T17N)对内皮细胞分化和迁移功能的影响．采用相差显微镜观察EBs发育和分化特征,Pull down分析Rac1表达变化,免疫荧光染色和Western blot分析内皮分化标志物,Matrigel凝胶实验观察血管索形成．结果表明,无论过表达或抑制Rac1的活化,并不影响EBs发育,均可形成典型的EBs胚层结构．抑制Rac1活化对内皮细胞系的发育无影响,但分化的内皮细胞不能连接成血管网．活化的Rac1表达减少,细胞迁移受到明显抑制．抑制Rac1活化导致细胞骨架F-actin排布紊乱．以上结果提示,Rac1影响胚胎早期血管发育的因素是抑制细胞游走,后者可能是通过F-actin机制所介导．     

问题解答(1)

问:绿色植物在有阳光时行光合作用吸二氧化碳放出氧气,在晚上没阳光时只有呼吸作用吸氧气放二氧化碳,为什么总说早晨到树林中散步空气好呢? 答:因为空气流动得很厉害,所以在一般树林中白天夜晚O_2及CO_2含量的改变是极小的.说早晨空气好,并不是植物起了什么作用,而是因为夜间人们的活动停止了,车马不在路上奔驰了,工厂的烟囱不冒烟了.加上树林中的湿度较大,尘土都降落在地上,空气就更为清洁了.我们夜间睡在屋中,屋中温度较高,不好气味的有机物(器物中发出或人呼出)空气中也很多,所以一到树林中,凉爽而清洁的空气,眼界的开朗就使我们心神为之一振.(吴相钰、董愚得答)     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

Rac1促进异质型细胞叠套结构的形成

异质型细胞叠套结构（heterotypic cell-in-cell structure，heCICs）与肿瘤发生和发展密切相关，在生命科学研究中的重要性逐渐显露。Ras相关C3肉毒毒素底物1（Ras-related C3 botulinum toxin substrate 1，Rac1）属于经典的Rho GTP酶，在细胞骨架以及细胞运动中起到关键调控作用。为研究Rac1在heCICs形成中的作用和机制，利用活细胞示踪剂cell-tracker分别标记肿瘤细胞和免疫杀伤细胞，建立heCICs模型。利用Rac1抑制剂NSC23766抑制Rac1活性后发现，肿瘤细胞与免疫杀伤细胞之间的heCICs形成率显著降低。通过分子克隆技术获得重组质粒pQCXIP-Rac1-EGFP，进行病毒包装感染肿瘤细胞获得Rac1过表达细胞系。进一步检测Rac1过表达对heCICs形成能力的影响，结果表明，Rac1表达水平升高后，heCICs形成率显著升高。本研究显示Rac1具有促进heCICs形成的作用，这为Rac1作为细胞叠套相关疾病的药物治疗靶点奠定了研究基础。     

Rac1和Cdc42在乳腺癌中的表达和临床意义

目的:研究Rac1和Cdc42在人乳腺癌中的表达及临床意义。方法:收集339例人乳腺癌组织样本,通过免疫组化的方法检测Rac1和Cdc42的表达情况,并分析其与乳腺癌临床病理学特征间的相关性。结果:Rac1和Cdc42在正常乳腺组织中几乎不表达,而在肿瘤组织的阳性表达率分别为35.9%和38.5%,均较正常乳腺组织显著升高,差异均具有统计学意义(P0.001和P0.05)。卡方检验分析表明,二者的表达与患者的年龄、肿瘤大小、组织分化程度、HER2状态无关(P0.05),而与TNM分期、淋巴结转移、肿瘤侵袭、ER状态和Ki-67表达有相(P0.05)。相关性分析表明,Rac1和Cdc42的表达与高TNM分期(r分别为0.443和0.295;P均0.001)、淋巴结转移阳性(r均为0.480和0.562;P均0.001)、肿瘤侵袭(r分别为0.412和0.440;P均0.001)、ER阴性表达(r分别为-0.517和-0.342;P均0.001)以及Ki-67高表达(r分别为0.338和0.454;P均0.001)呈正相关。结论:在乳腺癌组织中,Rac1和Cdc42作为癌基因表达增加,可能在乳腺癌恶性进程中发挥重要作用。     

檀香小G蛋白Rac1基因的克隆与功能分析

植物Rac蛋白属于小分子G蛋白ROP家族,广泛参与活性氧(ROS)产生、激素信号转导和组织形态建成。檀香(Santalum album Linn.)是著名的珍贵树种,为半寄生植物,其正常生长需要根部特化的吸器从其他寄主植物摄取营养物质。该研究基于全长转录组数据,采用RT-PCR方法克隆得到了1个檀香Rac基因,命名为SaRac1。结果表明:(1)SaRac1基因全长594 bp,编码197个氨基酸,分子量21.55 kD,理论等电点9.32,为亲水性蛋白。(2)进化分析显示,SaRac1蛋白和拟南芥(Arabidopsis thaliana)AtRac1~6、AtRac9和AtRac11蛋白同属于典型的植物RacⅠ家族蛋白。(3)结构预测显示,该蛋白在N端为保守的G结构域,蛋白C端具有CaaL保守基序。(4)原生质体亚细胞定位试验显示,SaRac1蛋白定位于细胞核和细胞质。(5)组织特异性表达分析显示,SaRac1基因在根和吸器中表达量最高,幼叶和茎中表达量较高,在成熟叶和老叶中表达量较低。(6)用寄生植物吸器诱导因子2,6-二甲氧基对苯醌(DMBQ)处理,发现SaRac1受到DMBQ的强烈诱导,表达量在4 h达到最高。研究推测,SaRac1基因受吸器诱导因子调控进而参与檀香吸器形成过程。