The endothelial monocyte-activating polypeptide II (EMAP II) is a substrate for caspase-7

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for leukocytes. The mature cytokine is formed in apoptotic cells by cleavage of the precursor proEMAP II. Here we show that caspase-7 is capable of cleaving proEMAP II in vitro. A proEMAP II mutant, in which the ASTD cleavage site was changed to the sequence ASTA, was not processed by caspase-7. The caspase-7-mediated generation and release of mature EMAP II may provide a mechanism for leukocyte recruitment to sites of programmed cell death, and thus may link apoptosis to inflammation.     

Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.     

Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein

The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases.     

Novobiocin induces the in vivo cleavage of active gene sequences in intact cells

When the topoisomerase II inhibitor, novobiocin, is administered to embryonic chicken red blood cells, it induces the in vivo release of an endogenous nuclease which cleaves specifically within internucleosomal spacer DNA and within nuclease-hypersensitive sites in the active chromatin of intact cells. This in vivo released nuclease activity is induced by novobiocin only in metabolically active immature red blood cells. Little induction occurs in mature erythrocytes and no induction occurs in cells previously treated with 2,4-dinitrophenol. Although novobiocin is required to induce release and/or activation of the nuclease, the activity of the nuclease, once activated, is independent of novobiocin. Analysis of the cleaved DNA in drug-treated immature cells demonstrates that the novobiocin-induced nuclease has an unusual blunt-ended double-stranded mode of cleavage. Because of its special properties and apparent chromatin related function in vivo, the novobiocin-induced nuclease activity offers a novel and useful in vivo and in vitro probe of chromatin structure.     

The efficacy of a novel, dual PI3K/mTOR inhibitor NVP-BEZ235 to enhance chemotherapy and antiangiogenic response in pancreatic cancer

Gemcitabine has limited clinical benefits for pancreatic ductal adenocarcinoma (PDAC). The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in PDAC. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival experiments were performed in murine xenografts. BEZ235 caused a decrease in phospho-AKT and phospho-mTOR expression in PDAC (AsPC-1), endothelial (HUVECs), and fibroblast (WI-38) cells. BEZ235 inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on proliferation inhibition were observed in the BEZ235-gemcitabine combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with gemcitabine and EMAP, induced apoptosis in AsPC-1, HUVECs, and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and gemcitabine monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235 + gemcitabine (30 days, P = 0.007), BEZ235 + EMAP (27 days, P = 0.02), gemcitabine + EMAP (31 days, P = 0.001), and BEZ235 + gemcitabine + EMAP (33 days, P = 0.004). BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of gemcitabine and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment.     

Interaction of the C-terminal domain of p43 and the alpha subunit of ATP synthase. Its functional implication in endothelial cell proliferation.

Human p43 is associated with macromolecular tRNA synthase complex and known as a precursor of endothelial monocyte-activating polypeptide II (EMAP II). Interestingly, p43 is also secreted to induce proinflammatory genes. Although p43 itself seems to be a cytokine working at physiological conditions, most of the functional studies have been obtained with its C-terminal equivalent, EMAP II. To gain an insight into the working mechanism of p43/EMAP II, we used EMAP II and searched for an interacting cell surface molecule. The level of EMAP II-binding molecule(s) was significantly increased in serum-starved tumor cells. Thus, the EMAP II-binding molecule was isolated from the membrane of the serum-starved CEM cell. The isolated protein was determined to be the alpha subunit of ATP synthase. The interaction of EMAP II and alpha-ATP synthase was confirmed by enzyme-linked immunosorbent assay and in vitro pull down assays and blocked with the antibodies raised against EMAP II and alpha-ATP synthase. The binding of EMAP II to the surface of serum-starved cells was inhibited in the presence of soluble alpha-ATP synthase. EMAP II inhibited the growth of endothelial cells, and this effect was relieved by soluble alpha-ATP synthase. Anti-alpha-ATP synthase antibody also showed an inhibitory effect on the proliferation of endothelial cells mimicking the activity of EMAP II. These results suggest the potential interaction of p43/EMAP II with alpha-ATP synthase and its role in the proliferation of endothelial cells.     

Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF.

The three members of the neurotrophin family (NGF, BDNF and NT-3) are synthesized as large precursor proteins which undergo proteolytic processing to yield biologically active, mature neurotrophic factors. We have used in vitro mutagenesis to examine the pro-region in the NGF precursor protein as a first step towards a general understanding of the role of propeptides in the biosynthesis of neurotrophins. Our results demonstrate that only two small domains within the NGF propeptide are required for the expression and secretion of properly processed and biologically active, recombinant mouse NGF in COS-7 cells. Domain I plays an important role in the expression of active NGF while domain II is involved in proteolytic processing. Both domains are partially conserved between the propeptides of NGF proteins isolated from different species as well as BDNF and NT-3.     

CLE Peptides in Plants: Proteolytic Processing,Structure-Activity Relationship,and Ligand-Receptor Interaction

Ligand-receptor signaling initiated by the CLAVATA3/ ENDOSPERM SURROUNDING REGION (CLE) family peptides is critical in regulating cell division and differentiation in meristematic tissues in plants. Biologically active CLE peptides are released from precursor proteins via proteolytic processing. The mature form of CLE ligands consists of 12–13 amino acids with several post-translational modifications. This review summarizes recent progress toward understanding the proteolytic activities that cleave precursor proteins to release CLE peptides, the molecular structure and function of mature CLE ligands, and interactions between CLE ligands and corresponding leucine-rich repeat (LRR) receptor-like kinases (RLKs).     

Evaluation of poly-mechanistic antiangiogenic combinations to enhance cytotoxic therapy response in pancreatic cancer

Gemcitabine (Gem) has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). The present study investigated combinations of gemcitabine with antiangiogenic agents of various mechanisms for PDAC, including bevacizumab (Bev), sunitinib (Su) and EMAP II. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo experiments were performed via murine xenografts. Inhibition of in vitro proliferation of AsPC-1 PDAC cells by gemcitabine (10 μM), bevacizumab (1 mg/ml), sunitinib (10 μM) and EMAP (10 μM) was 35, 22, 81 and 6 percent; combination of gemcitabine with bevacizumab, sunitinib or EMAP had no additive effects. In endothelial HUVECs, gemcitabine, bevacizumab, sunitinib and EMAP caused 70, 41, 86 and 67 percent inhibition, while combination of gemcitabine with bevacizumab, sunitinib or EMAP had additive effects. In WI-38 fibroblasts, gemcitabine, bevacizumab, sunitinib and EMAP caused 79, 58, 80 and 29 percent inhibition, with additive effects in combination as well. Net in vivo tumor growth inhibition in gemcitabine, bevacizumab, sunitinib and EMAP monotherapy was 43, 38, 94 and 46 percent; dual combinations of Gem+Bev, Gem+Su and Gem+EMAP led to 69, 99 and 64 percent inhibition. Combinations of more than one antiangiogenic agent with gemcitabine were generally more effective but not superior to Gem+Su. Intratumoral proliferation, apoptosis and microvessel density findings correlated with tumor growth inhibition data. Median animal survival was increased by gemcitabine (26 days) but not by bevacizumab, sunitinib or EMAP monotherapy compared to controls (19 days). Gemcitabine combinations with bevacizumab, sunitinib or EMAP improved survival to similar extent (36 or 37 days). Combinations of gemcitabine with Bev+EMAP (43 days) or with Bev+Su+EMAP (46 days) led to the maximum survival benefit observed. Combination of antiangiogenic agents improves gemcitabine response, with sunitinib inducing the strongest effect. These findings demonstrate advantages of combining multi-targeting agents with standard gemcitabine therapy for PDAC.     

Proteasomes and RARS modulate AIMP1/EMAP II secretion in human cancer cell lines

The aminoacyl t-RNA synthetase interacting multifunctional protein (AIMP1) is the precursor of the multifunctional inflammatory cytokine endothelial monocyte-activating polypeptide II (EMAP II). We previously demonstrated that AIMP1 secretion by pituitary adenomas is inversely correlated with tumor diameter and with RARS expression, suggesting that a high amount of RARS associated with AIMP1 might prevent the secretion of the latter cytokine. In this study, we investigated the role of RARS in modulating the secretion of AIMP1 in HeLa and MCF7 cell lines and investigated the possible role of the multicatalytic protease in the cleavage of AIMP1 to generate EMAP II. Our data show that RARS over-expression impairs AIMP1 secretion by both HeLa and MCF7 cells. Moreover, proteasome inhibition impairs AIMP1 cleavage to produce EMAP II. These data indicate that RARS over-expression associates with a reduced AIMP1 secretion and that the multicatalytic protease is involved in the generation of the mature cytokine, EMAP II.     

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium that is conserved in higher metazoans.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.     

Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II

Background

Endothelial-Monocyte Activating Polypeptide (EMAP II) is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.

Methodology/Principal Findings

Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.

Conclusions/Significance

Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.     

Change in the MGMT gene expression under the influence of exogenous cytokines in human cells in vitro

The influence of cytokines LIF, SCF, IL-3, and EMAP II and the Laferobion (IFN-a2b) drug on the MGMT gene expression in human cell cultures has been studied. It was shown that exogenous cytokines can modulate the MGMT gene expression at the protein level. EMAP II is able to increase or decrease the MGMT level, depending on the experimental conditions. Cytokines LIF, SCF, IL-3 and Laferobion decreased the MGMT expression level in human cells in vitro. Some conditions leading to the destruction of the MGMT protein complex were identified.     

Expression of human T-cell leukemia virus type I protease in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.     

Intracellular forms of Drosophila topoisomerase II detected with monoclonal antibodies.

We developed monoclonal antibodies against Drosophila topoisomerase II and studied the intracellular forms and the in vivo and in vitro proteolytic degradation of the enzyme. In purified enzyme preparations polyclonal sera and monoclonal antibodies recognized several polypeptides in the 170-132 kD molecular weight range. In vivo, however, the pattern was much simpler. In Drosophila embryos, pupae, fly heads and Schneider S3 tissue culture cells topoisomerase II appeared as a single 166 kD polypeptide. In Drosophila embryos, with two monoclonal antibodies topoisomerase II appeared as a doublet composed of the 166 kD canonical form and a slightly higher molecular weight polypeptide. Topoisomerase II was shown to be present also in fly heads which are composed entirely of nonproliferative tissues.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.