三丁基锡和微囊藻毒素对小鼠肝脏p-Ezrin的影响简

正埃兹蛋白(Ezrin)是连接细胞膜和细胞骨架之间的膜细胞骨架连接蛋白,通过其C端、N端分别与细胞骨架和细胞膜蛋白结合,属于ERM(Ezrin-radixin-moesin)家族成员。由于Ezrin在细胞中的特殊位置,不仅在细胞的运动、黏附等正常生理活动中起重要作用,而且能调控黏附分子,参与癌细胞信号转导。Ezrin在细胞中有活化和失活两种状态,目前认为主     

从皮层细胞骨架到信号分子:ERM蛋白质的调控魔力

埃兹蛋白(Ezrin)、根蛋白(Radixin)和膜突蛋白(Moesin)(ERM)广泛分布于细胞基质、微绒毛和粘着连接处。其N、C末端分别包含与膜蛋白、肌动蛋白相结合的位点,两端之间以α-螺旋区相连。ERM通过分子内N、C两端的相互作用而维持休眠状态,特异有序的信号作用能解除这种分子内作用使其活化。ERM介导着肌动蛋白-膜蛋白的连接并调控信号分子的传导,因此参与了细胞膜的组建、细胞黏附、细胞迁移,甚至肿瘤发展等生理过程。     

SCF和APC/C的结构及功能

细胞周期蛋白依赖性蛋白激酶(cyclin dependent kinases,CDKs)是细胞周期进行的推动力,泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)通过对细胞周期蛋白(cyclin)和CDK抑制物(CDK inhibitors,CKIs)的蛋白质水解作用来实现对CDKs活性的调控。SCF(Skp1-Cul1-F-box protein)和APC/C(anaphase-promoting complex/cyclosome)这两个泛素连接酶复合物参与了很多细胞周期调节因子的泛素化作用。它们参与的蛋白质降解系统的功能失调可能导致细胞增殖紊乱、基因组不稳定和肿瘤的发生。现对这两个泛素连接酶复合物的结构以及它们在细胞周期调控和肿瘤发生机制中的作用进行综述。     

钙黏附素和β- 连接素与宫颈上皮内瘤变进展的关系

目的:观察钙黏附素(E-cadherin)和β-连接素(β-catenin)在不同级别宫颈上皮内瘤变组织中的表达,探讨其与宫颈上皮内瘤变进展的关系。方法:采用免疫组化法检测51例宫颈组织病理存档石蜡包埋组织钙黏附素(E-cadherin)和β-连接素(β-catenin)的表达,阳性信号采用图像分析仪进行定量分析。结果:E-cadherin在柱状上皮移位组及CIN I组、CIN II组、CIN III组中的平均光密度值(AOD)分别为:0.0866±0.0392、0.073±0.0122、0.0467±0.0056、0.0396±0.0097;β-catenin在柱状上皮移位组及CIN I组、CIN II组、CIN III组中的平均光密度值(AOD)分别为:0.1101±0.0116、0.1016±0.0108、0.0711±0.0062、0.0515±0.0091。随着CIN病变的升级,钙黏附素和β-连接素的表达均呈下降趋势,二者的平均光密度值(AOD)正相关(r=2.546,P=0.018<0.05)。结论:钙黏附素、β-连接素与宫颈上皮内瘤变的进展相关,在估计CIN的预后中有一定意义。     

L-Periaxin蛋白与Ezrin蛋白相互作用初步分析

Periaxin是施旺(Schwann)细胞中特异表达的支架蛋白之一,参与髓鞘成熟及稳定. Periaxin的基因缺失或突变导致脱髓鞘型腓骨肌萎缩症(CMT)4F亚型的发生. 埃兹蛋白(ezrin)是一种膜骨架连接蛋白,其在维持细胞形态和运动方面具有重要作用,与肿瘤细胞转移密切相关. 本文通过免疫共沉淀、双荧光共定位、YFP双分子荧光互补、GST-pull-down等实验方法,分析了L-periaxin蛋白与ezrin蛋白之间的相互作用. 结果表明,L-periaxin蛋白与ezrin蛋白可在细胞质中共定位并可发生相互作用.     

α-亚麻酸-阿霉素前药的合成以及抗肿瘤活性研究

目的:合成具有酸敏特性的α-亚麻酸-阿霉素前体药物,并观察其抗肿瘤活性以及对肿瘤细胞的靶向性。方法:以BocNHNH2、α-亚麻酸、阿霉素为主要反应原料,先后合成Boc保护的α-亚麻酰肼、α-亚麻酰肼和α-亚麻酸-阿霉素腙键连接物,采用核磁共振、质谱等方法鉴定其结构;采用LC/MS方法研究连接物在不同pH介质中的药物释放行为;采用MTT法观察连接物对人肝癌细胞HepG2、乳腺癌细胞MDA-MB-231和MCF-7细胞的毒性作用。结果:在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)的催化作用下,合成了α-亚麻酸-阿霉素前体药物,波谱分析提示为目标产物。通过在体外酸性条件下水解连接物,我们发现该连接物具有显著的pH值敏感性。同时在体外细胞毒性实验中,我们发现相比于游离阿霉素,该合成连接物α-亚麻酸-阿霉素对HepG2、MDA-MB-231和MCF-7三种肿瘤细胞具有更强的细胞毒性,而且细胞毒性作用可被外源性α-亚麻酸抑制。结论:α-亚麻酸-阿霉素连接物能经α-亚麻酸受体介导靶向于α-亚麻酸受体丰富的肿瘤细胞,是一种潜在的新型抗肿瘤药物。     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

人源性抗TNFα小分子抗体的改构和分析

在获得人源性抗人TNF α单链抗体 (ScFv)基因序列的基础上 ,对ScFv的连接肽部分进行基因改造 ,并构建了Fab抗体基因。改构前后的ScFv分别重组入表达载体pBV2 2 0 ,经 42℃热诱导 ,在E .coliDH5α中表达了ScFv蛋白 ,得到分子量约为 30kD的重组蛋白质 ,改构前后ScFv的表达量分别占菌体总蛋白质的 6 .5 %和13 .8%。同时构建Fab可溶性表达载体并转化非抑制型菌株HB2 15 1,经IPTG诱导 ,在约 5 0kD分子量处呈现一条新生蛋白质条带。从大肠杆菌裂解液中对ScFv进行了复性和层析纯化 ,对Fab基因的表达产物进行了亲和层析纯化 ,并证实 :(1)改构后ScFv在大肠杆菌中的表达量有所提高 ;(2 )改构前后的ScFv与Fab均具有与hTNF α相结合的活性 ,具有GGGGS连接肽的ScFv与hTNF α的亲和常数为 6 .70× 10 4 (mol/L) -1,而改构后具有(GGGGS) 3 连接肽的ScFv的亲和常数提高为 7.2 7× 10 5(mol/L) -1,Fab与hTNF α的亲和常数为 7.6 1× 10 5(mol/L) -1,Fab与改构后ScFv的亲和力无明显差异 ;(3)ScFv与Fab均有中和hTNF α细胞毒的作用 ,具有(GGGGS) 3 连接肽的ScFv与Fab的中和活性基本相同 ,但均明显低于一株鼠源性单抗     

综述: 糖基转移酶超家族在肿瘤转移中的作用

蛋白质的糖基化修饰主要包括N-连接糖基化、O-连接糖基化和糖基磷脂酰肌醇锚定连接．与核酸和蛋白质不同,糖链的合成过程并不遵循传统的基因信息传递的中心法则,主要由一系列催化糖苷键形成的糖基转移酶完成．异常糖基化修饰被认为与恶性肿瘤的发生发展和临床预后密切相关．研究表明,糖基转移酶的表达及其糖链结构的异常可通过调节肿瘤细胞与细胞外基质的相互作用,继而影响肿瘤转移的关键步骤,如上皮间质转化(E-钙黏着蛋白、N-钙黏着蛋白)、细胞的移动性(整合素β1和α5)、侵袭(基质金属蛋白酶MMPs)、浸润(唾液酸化Lewis抗原sLeX和sLeA)．本文主要就唾液酰基转移酶、岩藻糖基转移酶和N-乙酰氨基葡萄糖转移酶等三大糖基转移酶家族的结构和生物学功能及其在肿瘤转移中的作用作一综述,以期为肿瘤转移的预测和诊断提供新思路．     

抗TNF-α/抗ED-B双特异抗体在酵母中的分泌表达与活性分析

赋予抗TNF-α单链抗体片段(TNF-sc Fv)对炎症组织的特异性,用一段来自人清蛋白(HSA)的柔性连接肽在基因水平上连接TNF-sc Fv和抗B型纤维连接蛋白(B-FN)的额外域B(ED-B)的sc Fv L19,构建了抗TNF-α/抗ED-B单链双特异抗体Bs Db,其中B-FN为炎症组织中特异表达的抗原。Bs Db在毕赤酵母中获得了分泌表达,表达产物经鉴定和纯化制备后,进行了功能分析。结果表明,Bs Db保留了其亲本抗体TNF-sc Fv和L19对抗原的免疫反应性,能够同时结合TNF-α和ED-B,并中和TNF-α的生理作用。而且,Bs Db对抗原的亲和力及中和能力与大肠杆菌包涵体来源的亲本抗体相比显著增强。在小鼠佐剂型关节炎(AIA)模型中,Bs Db能选择性地积累和保留于小鼠的炎症关节,并快速从血浆中清除。说明Bs Db兼备炎症组织的特异性和正常组织的低毒性,在类风湿关节炎及其他慢性炎症性疾病的治疗上具有较大潜力。     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.     

Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain

Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NH2-terminal domain (ezrin 1-233) and that of a COOH-terminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Sf9 cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 microns in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstrom, and A. Vaheri. 1994. J. Cell Biol. 126:1445- 1453), suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NH2- terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.     

Matriptase-2 inhibits HECV motility and tubule formation in vitro and tumour angiogenesis in vivo

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.     

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Characterization of the Ca2+ -regulated ezrin-S100P interaction and its role in tumor cell migration

Ezrin is a multidomain protein providing regulated membrane-cytoskeleton contacts that play a role in cell differentiation, adhesion, and migration. Within the cytosol of resting cells ezrin resides in an autoinhibited conformation in which the N- and C-terminal ezrin/radixin/moesin (ERM) association domains (ERMADs) interact with one another. Activation of the ezrin membrane-cytoskeleton linker function requires an opening of this interdomain association that can result from phosphatidylinositol 4,5-bisphosphate binding to the N-ERMAD and threonine 567 phosphorylation in the C-ERMAD. We have shown that ezrin can also be activated by Ca(2+)-dependent binding of the EF-hand protein S100P. We now provide a quantitative analysis of this interaction and map the respective binding sites to the F2 lobe in the ezrin N-ERMAD and a stretch of hydrophobic residues in the C-terminal extension of S100P. Phospholipid binding assays reveal that S100P and phosphatidylinositol 4,5-bisphosphate compete to some extent for at least partially overlapping binding sites in N-ERMAD. Using interaction-competent as well as interaction-incompetent S100P derivatives and permanently active ezrin mutants, we also show that the protein interaction and a resulting activation of ezrin promote the transendothelial migration of tumor cells. Thus, a prometastatic role of ezrin and S100P that had been proposed based on their overexpression in highly metastatic cancers is probably due to a direct interaction between the two proteins and the S100P-mediated activation of ezrin.     

Altered expression of ezrin in esophageal squamous cell carcinoma.

Ezrin is a membrane-cytoskeletal linker belonging to the ezrin-radixin-moesin (ERM) family and has been suggested to be involved in tumorigenesis. In this study we investigated ezrin expression pattern in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and the correlation with clinical characteristics. Immunohistochemical staining showed a tendency for ezrin to translocate from membrane to cytoplasm in the progression from normal epithelium to invasive carcinoma of the esophagus. By Western blot, we found that ezrin expression was downregulated in 13 ESCC specimens and upregulated in 36 others. Moreover, quantitative real-time RT-PCR demonstrated that ezrin mRNA level in normal esophageal mucosa was 3.60 +/- 3.60 times that in ESCC (p<0.001). Proliferating cell nuclear antigen (PCNA) expression level was higher in ezrin downregulated group compared with that in ezrin upregulated group (p<0.05). However, there was no significant association between ezrin expression and clinical characteristics. The results suggested that the localization of ezrin by immunohistochemistry may be useful in the diagnosis of ESCC, and ezrin may play a suppressive role in the tumorgenesis of ESCC.     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.     

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation

BackgroundEzrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear.MethodsDistribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis.ResultsEzrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo.ConclusionEzrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.