血管生成素相互作用蛋白质的筛选与鉴定

血管生成素（angiogenin, ANG）在肿瘤、神经退行性疾病和先天免疫过程中均发挥作用,但对其具体生理病理功能和作用机制的了解并不深入全面.蛋白质 蛋白质相互作用调控着细胞内的各个生物学过程,可以为目标蛋白质功能和机制的探索提供信息.本文利用酵母双杂交技术,分别从人心肌和肝cDNA文库中筛选了ANG的可能相互作用蛋白质.对筛选获得的20个候选蛋白质进行生物信息学分析,显示10个蛋白质含有EGF结构域;有5个蛋白质在KEGG 数据库已有记录,主要参与细胞黏附、通讯和迁移等过程.在以往的研究中,我们已经验证α 辅肌动蛋白2（α-actinin 2）、卵泡抑素（follistatin）、磷脂混杂酶1（phospholipid scramblase 1）和腓骨蛋白1（fibulin1）与ANG作用的真实性.本文的蛋白质沉降实验显示,ANG与腓骨蛋白2、3、4之间也存在相互作用.     

Alpha-actinin-2, a cytoskeletal protein, binds to angiogenin

Angiogenin is an angiogenic factor which is involved in tumorigenesis. However, no particular intracellular protein is known to interact directly with angiogenin. In the present study, we reported the identification of alpha-actinin-2, an actin-crosslinking protein, as a potential angiogenin-interacting partner by yeast two-hybrid screening. This interaction was confirmed by different approaches. First, angiogenin was pulled down together with His-tagged alpha-actinin-2 by Ni(2+)-agarose resins. Second, alpha-actinin-2 was coimmunoprecipitated with angiogenin by anti-angiogenin monoclonal antibody. Third, the in vivo interaction of these two proteins was revealed by fluorescence resonance energy transfer analysis. Since members of alpha-actinin family play pivotal roles in cell proliferation, migration, and invasion, the interaction between alpha-actinin-2 and angiogenin may underline one possible mechanism of angiogenin in angiogenesis. Our finding presents the first evidence of an interaction of a cytosolic protein with angiogenin, which might be a novel interference target for anti-angiogenesis and anti-tumor therapy.     

Identification and characterization of follistatin as a novel angiogenin-binding protein

Angiogenin enhances tumorigenesis. However, the mechanisms of angiogenin-induced angiogenesis and cancer cell proliferation remain elusive. In this study, follistatin was identified as a binding partner of angiogenin by a yeast two-hybrid screen and confirmed by a pull-down experiment. The interaction of fluorescently tagged angiogenin and follistatin was monitored in real time by a laser confocal microscope and shown to localize at the sub-nuclear region of HeLa cells. Additional yeast two-hybrid analysis revealed that domains 2 and 3 of follistatin were the minimal structure requirement for angiogenin binding. These findings provide new clues for further studies on the mechanisms of angiogenin-induced angiogenesis or cancer cell growth.     

p38 MAPK Down-regulates Fibulin 3 Expression through Methylation of Gene Regulatory Sequences: ROLE IN MIGRATION AND INVASION*

p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/β inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38β activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.     

血管生成素基因组结合序列的筛选与鉴定

血管生成素（angiogenin,ANG）调控细胞增殖、迁移、分化等生物学过程,但其作用的分子机制尚未完全明了. 目前认为,ANG可结合到rDNA区域促进rRNA转录,也可能与mRNA有结合.为全面鉴定细胞内可结合ANG的基因组序列,我们利用染色质免疫共沉淀结合DNA芯片技术（ChIP-chip）对HeLa细胞的基因组DNA进行了筛选,共获得了1 248个结合片段. 我们进一步分析了这些结合片段附近分布的基因,发现有699个可能受ANG结合调控的基因. 基因注释和聚类分析显示,这些可能受ANG调控的基因主要与肿瘤发生发展有关（特别是结直肠癌和前列腺癌）,并且与TGF-β和Wnt信号通路相关. 最后,我们验证了ANG不仅与WNT6、CCNE1、APC2、FZD8和EGFR基因的启动子区域有直接结合,而且调控其表达.以上研究结果为深入研究ANG的功能机制提供了线索.     

Angiogenin and its role in angiogenesis]

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique among them in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell-receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to a cell-surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin with angiogenin, stimulation of the cell-associated proteolytic activity by angiogenin; promotion of the cultured cells invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivo by angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Fibulin binds to itself and to the carboxyl-terminal heparin-binding region of fibronectin.

Fibulin is a recently described extracellular matrix (ECM) and plasma glycoprotein (Argraves, W. S., Tran, H., Burgess, W. H., and Dickerson, K. (1990) J. Cell Biol. 111, 3155-3164). In this report, ligand affinity chromatography and solid-phase binding analyses were performed to determine which ECM protein(s) interact with fibulin. Fibulin-Sepharose bound two polypeptides of 240 and 100 kDa from the culture medium of metabolically radiolabeled fibroblasts. These two proteins were identified as fibronectin (FN) and fibulin, respectively, based on their electrophoretic behavior and reactivity with monoclonal antibodies. Consistent with the findings of affinity chromatography, fibulin bound to surfaces coated with FN (either plasma or cellular form) or fibulin but not with other ECM proteins, such as laminin, merosin, and types I and IV collagen. The binding of fibulin to solid-phase FN was estimated to have a Kd of 139 nM, whereas the Kd for self-interaction was 322 nM. Evaluation of proteolytic fragments from all regions of FN allowed a fibulin-binding site to be localized within a 23-kDa heparin-binding fragment containing type III repeats 13-14. Heparin did not compete for the interaction between fibulin and FN, suggesting that the binding sites for fibulin and heparin are distinct.     

GON-1 and fibulin have antagonistic roles in control of organ shape

Most developing organs are surrounded by an extracellular matrix (ECM), which must be remodeled to accommodate growth and morphogenesis. In C. elegans, the GON-1 ADAMTS metalloprotease regulates both elongation and shape of the developing gonad . Here, we report that either human ADAMTS-4 or ADAMTS-9 can substitute for GON-1 in transgenic worms, suggesting functional conservation between human and nematode homologs. We further identify fibulin (FBL-1), a widely conserved ECM component , as critical for gonadal morphogenesis. FBL-1 is expressed in nongonadal tissues but is present at the surface of the elongating gonad. A fibulin deletion mutant has a wider than normal gonad as well as body size defects. We find that GON-1 and fibulin have antagonistic roles in controlling gonadal shape. Depletion of fbl-1, but not other ECM components, rescues gon-1 elongation defects, and removal of gon-1 rescues fbl-1 width defects. Therefore, the GON-1 protease normally promotes tissue elongation and expansion, whereas the fibulin ECM protein blocks these key morphogenetic processes. We suggest that control of organ shape by GON-1 and fibulin in C. elegans may provide a model for similar cellular processes, including vasculogenesis, in humans.     

Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证

通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ａng促血管新生的分子机制创造了条件.     

Fibulin 2, a Tyrosine O-Sulfated Protein,Is Up-regulated Following Retinal Detachment

Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition.     

Angiogenin depresses aortic smooth muscle cell cAMP by a pertussis toxin sensitive mechanism

Angiogenin transiently depresses the cAMP level of rat aortic smooth muscle cells. The dose response is similar to angiogenin activation of the inositol-specific phospholipase C in this cell line [Moore, F. & Riordan, J.F. (1989) Biochemistry. Submitted]. The time course showed a maximal depression (28%) in cAMP at 2 min, followed by a return to that of unstimulated cells by 3.5 min. Angiogenin also inhibited isoproterenol stimulated cAMP formation, but the percentage depression in cAMP (9%) was less than that in cells treated with angiogenin alone (28%). In contrast angiogenin enhanced forskolin stimulation of adenylate cyclase, an effect previously linked with agonist activation of protein kinase C. The effect of angiogenin on cellular cAMP was abolished by pre-incubation with pertussis toxin. Angiogenin had no effect on cellular cGMP. These results are consistent with activation of adenylate cyclase Gi following exposure of the cells to angiogenin and provide further evidence for interaction between cellular signalling pathways.     

Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

Background

In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.

Methodology/Principal Findings

In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.

Conclusions/Significance

Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.     

Overexpression of HTRA1 leads to ultrastructural changes in the elastic layer of Bruch's membrane via cleavage of extracellular matrix components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.     

Possible role of differentially expressing novel protein markers (ligatin and fibulin‐7) in human aqueous humor and trabecular meshwork tissue in glaucoma progression

The pathological mechanism underlying glaucoma has always been a complex aspect of this permanently blinding disease but proteomic studies have been helpful in elucidating it to a great extent in several studies. This study was designed to evaluate the expression and to get an idea about the function of two novel markers (ligatin and fibulin‐7) identified in human aqueous humor (hAH) in relation to glaucomatous progression. A significant increase in the protein content of glaucomatous hAH compared to that of non‐glaucomatous controls (NG‐Ctrls) was observed. Ligatin, fibulin‐7, and its proteolysis were revealed in hAH of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG) and NG‐Ctrls. Quantification confirmed no significant difference in expression of ligatin, whereas fibulin‐7 was significantly (P < 0.05) low in hAH of PACG in comparison to NG‐Ctrls and POAG. Importantly the immunohistochemical assay for both indicated their possible involvement in the maintenance of the appropriate structure of TM in vivo. Since oxidative stress is a major contributor to glaucomatous pathogenesis, in vitro analysis of nuclear and cytoplasmic fractions indicated intracellular changes in localization and expression of ligatin upon oxidative insult of human trabecular meshwork (TM) cells. While no such changes were found for fibulin‐7 expression. This was also corroborated with the immunocytochemical assay. Though a study with a small sample size, this is the first report which confirms the presence of ligatin and fibulin‐7 in hAH, quantified their differential expression, and indicated the possibility of their involvement in the maintenance of the TM structure.     

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Novel properties of the nucleolar targeting signal of human angiogenin

The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.