Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

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.     

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K_d, are 0.89 and 0.18 μM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.     

Angiogenin in Parkinson Disease Models: Role of Akt Phosphorylation and Evaluation of AAV-Mediated Angiogenin Expression in MPTP Treated Mice

The angiogenic factor, angiogenin, has been recently linked to both Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). We have recently shown that endogenous angiogenin levels are dramatically reduced in an alpha-synuclein mouse model of PD and that exogenous angiogenin protects against cell loss in neurotoxin-based cellular models of PD. Here, we extend our studies to examine whether activation of the prosurvival Akt pathway is required for angiogenin''s neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+), as observed in ALS models, and to test the effect of virally-mediated overexpression of angiogenin in an in vivo PD model. Using a dominant negative Akt construct, we demonstrate that inhibition of the Akt pathway does not reduce the protective effect of angiogenin against MPP+ toxicity in the dopaminergic SH-SY5Y cell line. Furthermore, an ALS-associated mutant of angiogenin, K40I, which fails to induce Akt phosphorylation, was similar to wildtype angiogenin in protection against MPP+. These results confirm previous work showing neuroprotective effects of angiogenin against MPP+, and indicate that Akt is not required for this protective effect. We also investigated whether adeno-associated viral serotype 2 (AAV2)-mediated overexpression of angiogenin protects against dopaminergic neuron loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. We found that angiogenin overexpression using this approach does not reduce the MPTP-induced degeneration of dopaminergic cells in the substantia nigra, nor limit the depletion of dopamine and its metabolites in the striatum. Together, these findings extend the evidence for protective effects of angiogenin in vitro, but also suggest that further study of in vivo models is required to translate these effects into meaningful therapies.     

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V₁ domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40–42) negatively affects the catalytic activity but also increases the angiogenic activity. ¹⁵N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.     

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.     

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.     

Angiogenin levels and ANG genotypes: dysregulation in amyotrophic lateral sclerosis

Objective

To determine whether 5 single nucleotide polymorphisms (SNPs) associate with ALS in 3 different populations. We also assessed the contribution of genotype to angiogenin levels in plasma and CSF.

Methods

Allelic association statistics were calculated for polymorphisms in the ANG gene in 859 patients and 1047 controls from Sweden, Ireland and Poland. Plasma, serum and CSF angiogenin levels were quantified and stratified according to genotypes across the ANG gene. The contribution of SNP genotypes to variance in circulating angiogenin levels was estimated in patients and controls.

Results

All SNPs showed association with ALS in the Irish group. The SNP rs17114699 replicated in the Swedish cohort. No SNP associated in the Polish cohort. Age- and sex-corrected circulating angiogenin levels were significantly lower in patients than in controls (p<0.001). An allele dose-dependent regulation of angiogenin levels was observed in controls. This regulation was attenuated in the ALS cohort. A significant positive correlation between CSF plasma angiogenin levels was present in controls and abolished in ALS.

Conclusions

ANG variants associate with ALS in the Irish and Swedish populations, but not in the Polish. There is evidence of dysregulation of angiogenin expression in plasma and CSF in sporadic ALS. Angiogenin expression is likely to be important in the pathogenesis of ALS.     

High level production of bovine angiogenin in E.?coli by an efficient refolding procedure

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.     

Genome-wide Identification and Quantitative Analysis of Cleaved tRNA Fragments Induced by Cellular Stress

Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA^Met. We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.     

Human Angiogenin Lacks Specific Antimicrobial Activity

The antimicrobial activities of commercially available human angiogenin were studied against two pathogens, namely, Candida albicans and Streptococcus pneumoniae. In contrast to the data published earlier, antimicrobial action of angiogenin was rather limited and comparable to that of bovine serum albumin.     

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.     

Interaction between angiogenin and fibulin 1: evidence and implication

Angiogenin is an angiogenic factor involved in tumorigenesis. However, the mechanism of angiogenin's action remains elusive. In the present study, we identified fibulin 1, an extracellular matrix and plasma glycoprotein, as an angiogenin-interacting molecule by yeast two-hybrid screening. This interaction was further confirmed by two different approaches. First, fibulin 1 was co-immunoprecipitated with angiogenin by anti-angiogenin monoclonal antibody in vitro , suggesting angiogenin binds with fibulin 1 directly. Then fluorescence resonance energy transfer analysis showed that fibulin 1 interacted with angiogenin in COS-7 cells, showing that the binding could occur in a cellular context. As fibulin 1 plays an important role in cell proliferation, migration, adhesion, and stabilizes new-forming blood vessel wall, the interaction between fibulin 1 and angiogenin might underline one possible mechanism of angiogenin in angiogenesis and/or tumorigenesis.     

Author index for volume 181, number 2

A phosphodiester, which comes into resonance at 0.4 ppm in the ³¹P nuclear magnetic resonance spectrum of intact muscles, has been isolated from the pectoralis muscle of chickens with hereditary muscular dystrophy by perchloric acid extraction, barium and alcoholic fractionation, and chromatographic isolation procedures. The compound,l-serine ethanolamine phosphodiester, whose presence is a characteristic of the diseased chicken muscle, has been characterized by ³¹P, ¹³C, and ¹H nuclear magnetic resonance as well as by chemical and chromatographic procedures.     

1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum

We report the ¹H, ¹³C, and ¹⁵N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, isolated from the acidophilic gram-negative facultatively anaerobic metal-reducing alphaproteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c ₂ and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on ¹³C- and ¹⁵N- labeled substrates.     

Copper stimulates proliferation of human endothelial cells under culture

Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO₄ in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of ¹²⁵I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326–335, 1998. © 1998 Wiley-Liss, Inc.     

1H, 13C and 15N resonance assignments for Binder of Arl2, BART

We report ¹H, ¹³C and ¹⁵N resonance assignments for Binder of Arl Two (BART), an effector of the small G protein Arl2. The BMRB accession code is 15914.     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.     

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10^?9 g/L), pigment epithelium growth factor (10^?8 g/L), hepatocyte growth factor activator (10^?7 g/L) and thrombospondin-1 (10^?6 g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.     

Angiogenin-Cleaved tRNA Halves Interact with Cytochrome c,Protecting Cells from Apoptosis during Osmotic Stress

Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5′ and 3′ tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.