氧化应激对Hsp90α、ARF1 细胞内定位和相互作用的影响

目的:探讨氧化应激对热休克蛋白90α(Hsp90α)与ADP-核糖基化因子1(ARF1)细胞内定位、相互作用的影响。方法:应用500μM H2O2处理HepG2细胞,建立氧化应激模型,MTT比色法检测细胞活力,Western blotting检测Hsp90α和ARF1水平,细胞免疫荧光法、免疫共沉淀检测上述蛋白在氧化应激下的分布、共定位变化和相互作用。结果:MTT比色法结果提示,随氧化应激时间延长,细胞存活力降低;Western blotting结果显示,氧化应激可提高胞内Hsp90α和ARF1蛋白水平;免疫共沉淀结果显示,随氧化应激作用时间延长,Hsp90α与ARF1相互结合增多;细胞免疫荧光结果显示,随氧化应激作用时间延长,Hsp90α与ARF1荧光强度增强,并趋于沿胞膜分布。结论:提示氧化应激影响Hsp90α和ARF1的水平、胞内分布及相互作用。     

Decomposition of Plant Debris by the Nematophagous Fungus ARF

In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive.     

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin^S66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.     

okemon及其在肿瘤中的研究进展

Pokemon 基因即POK 红系髓性致癌因子，也被称为FBI -1 ，LRF ，OCZF ，TIP ，它是POK 转录抑制物家族成员之一，其编 码的产物具有BTB / POZ 域和锌指结构,它是第一个被发现的可变阅读框基因ARF ( Alternative reading frame ，ARF )的特异性 转录抑制物，Pokemon 通过特异抑制ARF 转录来调控细胞周期、诱导肿瘤发生、促进肿瘤的发生发展，在一些人类肿瘤如食管鳞 癌、肺癌、结肠癌、人贲门癌、膀胱癌，前列腺癌和乳腺癌等肿瘤组织中具有较高的表达水平。Pokemon 的特殊性在于还能与其它 癌基因如Bcl-2，MDM2 等结合之后发挥作用来促进其他癌基因的活性。因此可以认为Pokemon 是肿瘤的总开关，对Pokemon 基 因的进一步研究有助于为肿瘤的诊断和治疗提供新的途径和方法。     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4,5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Cα as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

番茄ARF2蛋白的生物信息学分析与亚细胞定位

克隆番茄(Solanum lycopersicum) ARF2基因,并分析其分子特性和亚细胞定位,为研究其功能提供基础．通过生物信息学方法分析SlARF2基因编码蛋白的理化性质和分子特性．采用RT-PCR技术从番茄果实cDNA中扩增SlARF2基因全长,并构建与黄色荧光蛋白(YFP)融合的pBA-ARF2-YFP表达载体,进而再通过农杆菌介导的遗传转化方法,将重组质粒转化到野生型番茄中,将得到的T1代转基因种子萌发,然后取根尖通过荧光显微镜观察了融合蛋白在活细胞内分布的特点．生物信息学分析结果表明,SlARF2是富含Ser、Leu、Gly和Pro以及具有ARF家族典型结构域的可溶性蛋白,其氨基酸序列与葡萄、木薯和拟南芥的同源性分别为70.08 %、66.94 %和60.87 %．经酶切和测序分析证实pBA-ARF2-YFP融合表达载体构建成功,此外,PCR分析表明融合蛋白在转基因植株中得到表达．经荧光显微镜观察,ARF2定位在细胞核中．表明转录因子SlARF2定位在细胞核中,对番茄果实发育和成熟起重要作用．     

Interaction of p14ARF with Brca1 in cancer cell lines and primary breast cancer

We report an association between p14ARF and Brca1 in which both proteins co-immunoprecipitate (co-IP) in DU145 cells. The N-terminal 64 residues of p14ARF encoded by exon 1beta are sufficient for this association. Inside the cell, ectopic p14ARF co-localizes with ectopic and endogenous Brca1 in A375 cells. Endogenous p14ARF co-localizes with endogenous Brca1 in DU145 cells but not in H1299 cells. Since p14ARF interacts with B23 in the nucleolus, Brca1 co-localizes with B23 in DU145 but not in H1299 cells. While ectopic ARF potently inhibited DU145 cell proliferation, it had no effect on the proliferation of H1299 cells, suggesting that the interaction between ARF and Brca1 contributes to ARF-mediated tumor suppression. Consistent with this notion, ectopic p14ARF modulates endogenous Brca1 expression in MCF7 breast cancer cells and p14ARF co-localizes with Brca1 in normal breast epithelial cells. This co-localization is enhanced in primary breast cancer. Taken together, the results show that p14ARF associates with Brca1, which may play a major role in tumor suppression.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.