LIM蛋白家族的研究进展

LIM蛋白是分子结构中含有一个或多个LIM结构域的蛋白质家族,该家族中的蛋白质通过其LIM结构域与某些结构蛋白,激酶,转录调控因子等多种蛋白质相互作用,对某些基因的表达,细胞分化与发育,细胞骨架形成等发挥重要调控作用。本文介绍LIM蛋白家族的分类与功能,LIM蛋白及其与其他蛋白之间的相互作用,以及LIM蛋白在心血管系统中的作用。     

Gsα调控小鼠大脑皮层发育

异源三聚体G蛋白激活alpha亚基(Gsα),是一种普遍存在的鸟苷酸结合蛋白,调节受体介导的胞内cAMP信号通路进而参与调控细胞的生命活动。目前Gsα信号通路的研究主要在生物化学和药物方面,但在小鼠大脑皮层发育中的作用还没有详尽的描述。本研究首先利用cre-loxP系统在小鼠大脑皮层神经前体细胞中成功敲除Gsα基因;其次,通过收集出生后不同天数的正常小鼠和敲除小鼠,统计分析后发现敲除鼠的脑重和体重减轻;最后,对小鼠大脑做切片后染色,发现在小鼠大脑皮层条件性敲除Gsα基因的成年鼠中表达thy1-EGFP(增强绿色荧光蛋白)的神经元的数量减少,皮层形成异常。由此推测,Gsα在小鼠大脑皮层发育中发挥重要作用。     

Bmal1对小鼠胚胎期皮层神经元放射状迁移和轴突投射的影响

大脑皮层的发育是脑结构形成与功能建立的重要基础,在此过程中,皮层神经元放射状迁移及胼胝体区的轴突投射是必不可少的关键环节,该环节受基因转录的调控,但相关的分子机制目前仍不明确。转录因子BMAL1 (brain and muscle Arnt-like protein1)是体内重要的生物钟节律因子之一,最新研究发现其还参与调节海马神经祖细胞增殖,提示其与神经发育存在潜在的相关性。为明确Bmal1基因在大脑皮层发育中的具体作用,本研究首先通过RT-PCR和Real-timePCR检测Bmal1基因在神经系统中的表达情况。结果表明,Bmal1基因在神经系统中表达丰富,并且在发育期的大脑内呈现特定的表达规律:在胚胎后期和出生后早期脑内表达水平相对较高,以出生后第3 d为高峰。进一步通过联合使用小鼠子宫内胚胎电转和RNAi干扰方法敲减脑内神经元中Bmal1的表达水平,结果发现胚胎期皮层神经元的放射状迁移发生了延迟,延迟程度与RNAi的敲减效率呈正相关,存在一定的基因剂量-效应关系。进一步观察发现,在胚胎期脑内神经元中降低Bmal1表达水平以后,胼胝体轴突向对侧大脑半球的投射也出现了明显的缺陷。上述研究结果表明,BMAL1是大脑皮层神经元的放射状迁移以及轴突投射发育过程中的一个重要的调控分子,为从转录因子角度深入理解大脑皮层发育的分子调节机制和寻找调控靶点提供了新的线索。     

细胞骨架蛋白PDLIM1在肿瘤中的研究进展

人PDZ和LIM域蛋白1(PDZ and LIM domain protein 1, PDLIM1)是PDZ-LIM蛋白家族的成员之一,参与多种生物学过程,包括细胞骨架组织和肿瘤发生。PDLIM1通过PDZ结构域结合相关蛋白质(如辅肌动蛋白α-actinin)以及通过LIM结构域与相关激酶(如clik1)结合,并定位到肌动蛋白应力纤维,进而参与细胞骨架调控。细胞骨架具有维持细胞形态的功能,在细胞运动中起着关键作用。肿瘤细胞的浸润与转移常表现为细胞运动能力的改变,这个改变过程往往涉及到细胞骨架在时空上的动态重组,而重组的时空调控由关键的细胞信号通路介导。因此,进一步研究PDLIM1在肿瘤浸润与转移过程中的信号调控机制,可能发现潜在的治疗靶点和预后因子,有助于在精准医疗时代进行更好的个性化肿瘤防治。     

SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力

【目的】探究铜绿假单胞菌(Pseudomonas aeruginosa)鸟苷酸环化酶(diguanylate cyclase,DGC)SadC合成的环二鸟苷酸(cyclicdi-GMP,c-di-GMP)信号与PilZ结构域受体间的信号传递关系,分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析;SadC过表达背景下,PilZ结构域受体突变各菌株的泳动表型分析和筛选;基因敲除和过表达解析筛选出的PilZ结构域受体功能;定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力;PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制;功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强,而过表达PilZ或FlgZ则抑制了泳动能力;定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要,多序列比对分析表明这些位点是其保...     

DISC1基因及其编码蛋白的结构、功能与精神分裂症的相关研究进展

人染色体t(1;11)(q42.1;q14.3)的平衡易位可能导致Disrupted-in-Schizophrenia 1(DISC1)基因断裂,这一突变被认为与精神分裂症高度相关。DISC1属于细胞骨架蛋白,可能与超过200种蛋白相互作用,参与神经元分化、迁移、突触形成及可塑性等,对早期神经系统发育和成年期神经元功能产生重要影响。本文将就近期DISC1的研究进展进行综述。     

膜结合鸟苷酸激酶蛋白质家族的结构与功能

MAGUKs蛋白家族成员位于细胞表面,通过其PDZ、SH₃、GK结构域调节离子通道受体的聚集,构建细胞骨架,参与信号转导,调控细胞周期进程,并与机体神经发育密切相关.     

Erbin:LAP家族的新成员

Erbin(ErbB2结构蛋白）是LAP家族新成员,它的N末端有16个富含亮氨酸的重复序列（LRR）,在LRR后有一个LRR样的结构域,C末端含有PDZ结构域,实验证明Erbin通过其PDZ结构域与ErbB2结合。在上皮和神经元等级性细胞中,Erbin对ErbB2的定位或定向转移具有重要作用,并可增加ErbB2在细胞表面的表达量。Erbin作为支架蛋白还参与了细胞骨架的形成。     

Reelin信号通路在大脑新皮质形成中的作用

人类大脑皮层发育过程中,大量神经细胞从靠近脑室的增殖区产生并向软脑膜方向迁移,最终形成与人类许多高级功能相关的6层大脑新皮质结构,Reelin-Dab1信号通路在神经细胞迁移中起到了关键的作用。Reelin结合于迁移细胞膜上的极低密度脂蛋白受体(very lowlipoprotein recep-tor,VLDLR)和载脂蛋白E受体2(apolipoprotein E receptor2,ApoER2),磷酸化胞浆内的衔接蛋白disabled1(Dab1),继而与下游信号分子如细胞骨架蛋白等相互作用,指导皮层神经元的正确迁移和定位。本篇综述讨论了Reelin-Dab1信号通路在人类新皮质形成过程中的作用及近几年的研究新进展。     

神经发育过程中的基因表达调控机制以及相关的神经发育性疾病（英文）

在中枢神经系统的发育过程中,内在的基因和外在的环境因素相互作用以确保神经元发育的各个阶段(如神经细胞的增殖、分化、迁移,轴突延伸,树突成长,功能性突触的形成等)有序进行。这一过程需要众多的基因表达调控机制对不同基因的表达水平进行精确的时空调节。这些调控机制包括了序列特异性DNA结合蛋白(转录因子等)、组蛋白修饰、DNA甲基化、以及微小RNA(mi RNA)等。它们形成了一个调控网络,在神经发育的不同阶段以及不同的环境刺激因素的情况下,从染色质的结构、基因的转录和蛋白质的翻译等不同层次上实现基因表达的精确调控。神经元发育过程中基因表达失调与一些神经发育性疾病相关,例如自闭症谱系障碍,Rett综合征,脆性X综合征以及其他遗传性疾病。深入研究神经元发育过程中基因表达调控机制可望能够给这些神经发育性疾病的诊断和治疗提供新的思路。     

Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.     

Requirement of the basic region of N-WASP/WAVE2 for actin-based motility

WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Delta basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Delta basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex.     

Focal adhesion kinase regulation of N-WASP subcellular localization and function

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.     

The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs

A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, we still lack the exact nature of its binding site. We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin.     

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.     

Neuronal specific βPix-b stimulates actin-dependent processes via the interaction between its PRD and WH1 domain of N-WASP

βPix, a Pak-interacting nucleotide exchange factor (Cool-1/p85SPR), is a Cdc42/Rac1-specific guanine nucleotide exchange factor (GEF) involved in various actin-related processes. Many previous studies have focused on ubiquitously expressed βPix-a, while the role of the neuronal-specific isoform βPix-b is still unknown, especially whether its role is distinct from or similar to βPix-a. Here we show that unlike βPix-a, overexpression of βPix-b stimulates actin-dependent comet formation in BHK21 cells. This effect is attributed to the interaction between its proline-rich domain (PRD) and the WH1 domain of N-WASP. In addition, we show that overexpression of βPix-b stimulates actin-dependent dendritic spine formation in rat hippocampal neurons in culture, a formation that is blocked by co-expression of the WH1 domain of N-WASP or the PRD of βPix-b. Knocking-down endogenous expression of βPix-b by shRNA reduced the number of dendritic spines, which were rescued only by PRD-containing βPix-b mutants. GEF activity of βPix-b is also required for these effects. The results show that neuronal-specific βPix-b stimulates actin-dependent processes in cells via the interaction between its PRD and the WH1 domain of N-WASP. Our results identify N-WASP as the first protein shown to interact with the PRD of βPix-b, raising the possibility that, as an N-WASP WH1-binding protein, βPix-b may regulate N-WASP's activity in cells.     

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.     

Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.     

Wsp1, a GBD/CRIB domain-containing WASP homolog, is required for growth, morphogenesis, and virulence of Cryptococcus neoformans

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.