Flotillin-1的生理功能及其在疾病中的作用研究进展

Flotillin-1(浮舰蛋白-1)是脂筏的标记蛋白。它不但参与多种细胞正常的生理活动,也在许多肿瘤发生和发展中起重要作用。近年发现Flotillin-1在肺腺癌、乳腺癌、肝细胞癌等细胞中高表达,并与临床等级、预后差、总体存活率低有关联。简要综述了Flotillin的组成、结构和基本功能,以及在疾病中的作用。     

窖蛋白-1与乳腺癌

 窖蛋白(caveolin)是分子量为21～24 kD的整合膜蛋白,是胞膜窖(caveolae)的标志性结构分子.目前已克隆并鉴定出窖蛋白基因家族的3个成员：窖蛋白-1,窖蛋白-2和窖蛋白-3.其中窖蛋白-1参与细胞内的许多生命活动,如胆固醇的运输,细胞膜的组装,细胞信号传导,细胞周期调控,细胞转化和肿瘤形成.窖蛋白-1还可以与转录因子相互作用,调节相关基因的表达,抑制肿瘤发生.另外,在乳腺癌、前列腺癌、胃癌、肝癌等多种恶性肿瘤中均发现窖蛋白-1的异常;近年来发现,窖蛋白-1与乳腺上皮细胞转化和乳腺癌发生密切相关.本文概括介绍了窖蛋白-1的结构特点、窖蛋白-1介导的信号通路及与乳腺癌发生的关系方面的研究进展.     

U937细胞中GATA1调控c-myb基因表达

c-myb是血细胞生成过程中的一个重要转录因子,与造血干细胞的增殖、分化、凋亡有关.在白血病、结肠癌、乳腺癌和黑色素瘤等恶性肿瘤中,c-myb异常表达,但是在白血病细胞中c-myb调控的机制尚不清楚.本研究探究了U937细胞中GATA1与c-myb的调控关系,可能对白血病研究和治疗提供帮助.用12-氧-十四烷酰佛波醇-13-乙酸酯(TPA)诱导U937细胞分化,并检测分化前后GATA1与c-myb蛋白的变化.Western blot结果显示U937细胞经TPA诱导分化后c-myb与GATA1蛋白均明显下降.利用慢病毒包装GATAl shRNA质粒和GATA1过表达质粒并感染到U937细胞中实现转录因子GATA1的敲降及过表达后,检测c-myb的mRNA和蛋白的表达水平.结果 显示:敲降GATA1后,c-myb的mRNA和蛋白质水平明显下降;过表达GATA1后,c-myb的mRNA和蛋白质水平明显上调.本研究揭示了U937细胞中GATA1对c-myb的正向调控关系,为白血病研究和治疗方案提供了新的思路.     

浮舰蛋白flotillins在细胞生理和疾病中的作用

浮舰蛋白flotillin-1和flotillin-2参与多个细胞生理活动过程,包括调节细胞黏附、物质内吞、蛋白质分选与再循环和细胞迁移,同时也与一些退行性疾病和肿瘤的发生发展有密切联系.该文介绍了flotillins的结构与定位,综述了flotillins从发现至今影响个体形态发生、神经元生长和功能以及调控多个信号转...     

YB-1与EMT对肿瘤发生与治疗影响的研究进展

正Y-box结合蛋白1(Y-box binding protein 1,YB-1)属于冷休克结构域(cold shock domain,CSD)家族中的一员,在原核及真核生物细胞中普遍存在,是一种与肿瘤生长、增殖及耐药性密切相关的多功能蛋白[1~3]。大量研究证实,YB-1在多种恶性肿瘤中过表达,如膀胱癌、宫颈癌及前列腺癌[4~6]等。上皮-间质转化(epithelial-mesenchymal transi-     

大黄鱼Flotillin-1基因分子特征分析

浮舰蛋白-1(Flotillin-1)是属于SPFH家族的蛋白,是重要的脂筏标志性蛋白.在构建大黄鱼(Larimichthys crocea)肌肉组织cDNA文库的基础上,克隆了Flotillin-1基因,并进一步扩增出内含子.克隆到的序列全长为2497 bp,其中编码区1194 bp,编码397个氨基酸.生物信息学分析大黄鱼Flotillin-1有5类20个功能位点,存在2次跨膜结构,N端和C端都位于细胞膜内.大黄鱼Flotillin-1氨基酸序列具有非常高的保守性,与大西洋鲑和斑马鱼的同源性都在80%以上.在组织中的表达也非常广泛,其中在脾中的表达最强.     

窖蛋白-1、基质金属蛋白酶-2与乳腺肿瘤的侵袭和转移

窖蛋白(caveolin)是分子量为21～24 kD的整合膜蛋白,是胞膜窖(caveolae)的标志性结构分子,其家族成员窖蛋白-1(caveolin-1,Cav-1)参与细胞内许多重要的生命活动.近来研究发现,窖蛋白-1与乳腺上皮细胞转化及乳腺癌的发生密切相关.基质金属蛋白酶(matrix metalloproteinases,MMPs)是基质降解代谢的主要酶类,几乎能降解细胞外基质和基底膜的所有成分,其家族成员明胶酶A(MMP-2)在乳腺癌的浸润和转移过程中起重要作用.新近发现,窖蛋白-1与基质金属蛋白酶-2在胞膜窖中共定位,窖蛋白-1通过抑制基质金属蛋白酶-2的激活来抑制乳腺癌的侵袭和转移,起到肿瘤抑制因子的作用.本文对窖蛋白-1与基质金属蛋白酶-2各自在乳腺肿瘤侵袭转移中的作用及两者关系的研究进行综述.     

膜联蛋白A1及其与肿瘤关系的研究进展

膜联蛋白A1 (Annexin A1,ANXA1)是一种来源于脊柱(哺乳)动物的钙依赖性磷脂结合蛋白,是介导细胞内糖皮质激素抗炎作用的效应分子,在组织中广泛表达,参与细胞生长周期的各个阶段.其既可以可溶性形式存在,也可稳定或可逆结合于细胞骨架蛋白,调控细胞与细胞外基质的相互作用.大量的研究发现,AnnexinA1的表达在不同肿瘤组织中有差异,并且同一肿瘤不同类型中表达也不一样,其异常表达及细胞内定位改变可能跟多种恶性肿瘤的分化及转移相关.Annexin A1与肿瘤的密切关系,或许可使其发展为一个新的肿瘤标志物,为肿瘤的早期诊断、治疗及预后提供新的判断标准.因此,探讨Annexin A1与肿瘤的关系极具临床应用前景.     

原代培养脊髓神经元线状溶酶体与细胞骨架蛋白-纽蛋白关系的研究

目的研究原代培养脊髓神经元线状溶酶体(nematolysosome)的形成与分布及其与细胞骨架蛋白-纽蛋白(vinculin)的关系.方法用细胞松弛素D(cytochalasin D,CD)及佛波醇酯(phorbol myristate acetate,PMA)处理原代培养脊髓神经元,用免疫荧光双标记纽蛋白及组织蛋白酶D(cathepsin D)、酸性磷酸酶(ACPase)、电镜细胞化学及共焦激光扫描显微镜方法研究线状溶酶体与纽蛋白的关系.结果在正常对照组神经元,组织蛋白酶D(标记溶酶体)与纽蛋白分布于胞质及突起内;在CD及PMA处理神经元,纽蛋白及组织蛋白酶D的分布呈向心性移动,但集聚的部位不同;电镜酶细胞化学方法显示CD组及PMA组神经元内线状溶酶体均增多.结论组织蛋白酶D及纽蛋白在培养脊髓神经元内协同分布,CD及PMA均可引起二者分布的变化,提示纽蛋白可通过增强细胞内吞体/溶酶体系统活动而使线状溶酶体增加,也可通过促进丝状肌动蛋白聚合而影响线状溶酶体的形成及运动.     

TWIST1蛋白与肿瘤北大核心CSCD

Twist1(人类直系同源物为TWIST1)蛋白是一种进化上高度保守的碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH)转录因子,最初在果蝇胚胎发育过程中发现其具有重要作用,Twist1基因突变引起头面部和肢体畸形。近年来研究表明,Twist1在多种恶性肿瘤中高表达,具有诱导上皮-间质转化,促进细胞迁移、侵袭,增强化疗药物抗性等生物学功能。本文主要对Twist1蛋白的结构特征、基因表达调控与蛋白修饰调控机制及其在肿瘤发生发展中的作用进行综述,为其作为潜在的药物靶点及癌症治疗研究提供新的启示。     

Evidence for Chemokine-mediated Coalescence of Preformed Flotillin Hetero-oligomers in Human T-cells

We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also targeted to the uropod of T-cells and neutrophils. Biochemical studies suggest formation of flotillin homo- and hetero-oligomers in other cell types, but so far knowledge is lacking on in situ flotillin organization in leukocytes. We have now analyzed flotillin organization in human T-cells using fluorescence resonance energy transfer (FRET). Coexpressed C-terminally tagged flotillin-1-mCherry and flotillin-2-enhanced green fluorescent protein (EGFP) show significant FRET when analyzed in intact human T-cells in the absence and presence of chemokine. In contrast, little FRET was observed between coexpressed flotillin-1-mCherry and flotillin-1-EGFP before or after chemokine addition, indicating predominant formation of heterodimers and/or -oligomers. Interestingly coexpression of untagged flotillin-2 strongly enhanced FRET between differently tagged flotillin-1 molecules in resting and chemokine-stimulated cells, indicating that close contacts of flotillin-1 molecules only occur in flotillin-2-containing hetero-oligomers. Comparable results were obtained for tagged flotillin-2. We further show that disruption of the actin network, depletion of intracellular calcium, and inhibition of phospholipase C all result in suppression of chemokine-induced polarization and flotillin cap formation, but do not abolish FRET between tagged flotillin-1 and -2. Our results support predominant formation of flotillin-1 and -2 hetero-oligomers in resting and chemokine-stimulated human T-cells which may importantly contribute to structuring of the uropod.     

PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts

PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.     

Variations of expression of lipid raft protein flotillin-2 in human lung adenocarcinomas and its influence on the characteristics of the lung cancer cell line A549

Transduction of many cellular signals is mediated by special plasma membrane microdomains that are called lipid rafts. Lipid rafts are dynamic and transient structures; however, they can be stabilized by lipid raft proteins, including the family of flotillins which is represented by flotillin-1 and flotillin-2. Flotillins are expressed in different tissues and may regulate many signalling pathways. However, their role in carcinogenesis remains unclear. The aim of this work was to investigate variations of flotillin-2 expression on mRNA and protein level in lung adenocarcinoma specimens. We also studied the influence of flotillin-2 expression on the characteristics of A549 lung cancer cell line. The level of flotillin-2 mRNA was reduced in the vast majority of investigated adenocarcinoma specimens in comparison to corresponding normal tissues. However, the amount of protein varied widely and was preferentially increased (40%) than decreased (15%). Flotillin-2 overexpression in A549 cell line did not change proliferation but stimulated migration of cultivated cells. Conversely, knockdown of flotillin-2 using small hairpin RNA (shRNA) downregulated proliferation as well as migration of tumor cells. These results indicate that the expression of flotillin-2 changes in human lung adenocarcinoma and that this protein may influence the key characteristics of tumor cells.     

Flotillin depletion affects ErbB protein levels in different human breast cancer cells

The ErbB3 receptor is an important regulator of cell growth and carcinogenesis. Among breast cancer patients, up to 50–70% have ErbB3 overexpression and 20–30% show overexpressed or amplified ErbB2. ErbB3 has also been implicated in the development of resistance to several drugs used against cancers driven by ErbB1 or ErbB2. One of the main challenges in ErbB-targeting therapy is to inactivate signaling mediated by ErbB2–ErbB3 oncogenic receptor complexes. We analyzed the regulatory role of flotillins on ErbB3 levels and ErbB2–ErbB3 complexes in SKBR3, MCF7 and MDA-MB-134-VI human breast cancer cells. Recently, we described a mechanism for interfering with ErbB2 signaling in breast cancer and demonstrated a molecular complex of flotillin scaffolding proteins with ErbB2 and Hsp90. In the present study, flotillins were found to be in a molecular complex with ErbB3, even in cells without the presence of ErbB2 or other ErbB receptors. Depletion of either flotillin-1 or flotillin-2 resulted in downregulation of ErbB3 and a selective reduction of ErbB2–ErbB3 receptor complexes. Moreover, flotillin-2 depletion resulted in reduced activation of Akt and MAPK signaling cascades, and as a functional consequence of flotillin depletion, breast cancer cells showed an impaired cell migration.     

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.     

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.     

Flotillins Directly Interact with γ-Catenin and Regulate Epithelial Cell-Cell Adhesion

Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6–8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.