Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 simultaneously binds Glutamyl-Prolyl-tRNA synthetase and scaffold protein aminoacyl tRNA synthetase complex interacting multifunctional protein 3 of the multi-tRNA synthetase complex

Higher eukaryotes have developed extensive compartmentalization of amino acid (aa) - tRNA coupling through the formation of a multi-synthetase complex (MSC) that is composed of eight aa-tRNA synthetases (ARS) and three scaffold proteins: aminoacyl tRNA synthetase complex interacting multifunctional proteins (AIMP1, 2 and 3). Lower eukaryotes have a much smaller complex while yeast MSC consists of only two ARS (MetRS and GluRS) and one ARS cofactor 1 protein, Arc1p (Simos et al., 1996), the homolog of the mammalian AIMP1. Arc1p is reported to form a tripartite complex with GluRS and MetRS through association of the N-terminus GST-like domains (GST-L) of the three proteins (Koehler et al., 2013). Mammalian AIMP1 has no GST-L domain corresponding to Arc1p N-terminus. Instead, AIMP3, another scaffold protein of 18 kDa composed entirely of a GST-L domain, interacts with Methionyl-tRNA synthetase (MARS) (Quevillon et al., 1999) and Glutamyl-Prolyl-tRNA Synthetase (EPRS) (Cho et al., 2015). Here we report two new interactions between MSC members: AIMP1 binds to EPRS and AIMP1 binds to AIMP3. Interestingly, the interaction between AIMP1 and AIMP3 complex makes it the functional equivalent of a single Arc1p polypeptide in yeast. This interaction is not mapped to AIMP1 N-terminal coiled-coil domain, but rather requires an intact tertiary structure of the entire protein. Since AIMP1 also interacts with AIMP2, all three proteins appear to compose a core docking structure for the eight ARS in the MSC complex

Therapeutic efficacy of anti-MMP9 antibody in combination with nab-paclitaxel-based chemotherapy in pre-clinical models of pancreatic cancer

Matrix metalloproteinase 9 (MMP9) is involved in the proteolysis of extracellular proteins and plays a critical role in pancreatic ductal adenocarcinoma (PDAC) progression, invasion and metastasis. The therapeutic potential of an anti-MMP9 antibody (αMMP9) was evaluated in combination with nab-paclitaxel (NPT)-based standard cytotoxic therapy in pre-clinical models of PDAC. Tumour progression and survival studies were performed in NOD/SCID mice. The mechanistic evaluation involved RNA-Seq, Luminex, IHC and Immunoblot analyses of tumour samples. Median animal survival compared to controls was significantly increased after 2-week therapy with NPT (59%), Gem (29%) and NPT+Gem (76%). Addition of αMMP9 antibody exhibited further extension in survival: NPT+αMMP9 (76%), Gem+αMMP9 (47%) and NPT+Gem+αMMP9 (94%). Six-week maintenance therapy revealed that median animal survival was significantly increased after NPT+Gem (186%) and further improved by the addition of αMMP9 antibody (218%). Qualitative assessment of mice exhibited that αMMP9 therapy led to a reduction in jaundice, bloody ascites and metastatic burden. Anti-MMP9 antibody increased the levels of tumour-associated IL-28 (1.5-fold) and decreased stromal markers (collagen I, αSMA) and the EMT marker vimentin. Subcutaneous tumours revealed low but detectable levels of MMP9 in all therapy groups but no difference in MMP9 expression. Anti-MMP9 antibody monotherapy resulted in more gene expression changes in the mouse stroma compared to the human tumour compartment. These findings suggest that anti-MMP9 antibody can exert specific stroma-directed effects that could be exploited in combination with currently used cytotoxics to improve clinical PDAC therapy

Vascular mediators in chronic lung disease of infancy: role of endothelial monocyte activating polypeptide II (EMAP II)

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Over the years, the BPD phenotype has evolved, but despite various advances in neonatal management approaches, the reduction in the BPD burden is minimal. With the advent of surfactant, glucocorticoids, and new ventilation strategies, BPD has evolved from a disease of structural injury into a new BPD, marked by an arrest in alveolar growth in the lungs of extremely premature infants. This deficient alveolar growth has been associated with a diminution of pulmonary vasculature. Several investigators have described the epithelial / vascular co-dependency and the significant role of crosstalk between vessel formation, alveologenesis, and lung dysplasia's; hence identification and study of factors that regulate pulmonary vascular emergence and inflammation has become crucial in devising effective therapeutic approaches for this debilitating condition. The potent antiangiogenic and proinflammatory protein Endothelial Monocyte Activating Polypeptide II (EMAP II) has been described as a mediator of pulmonary vascular and alveolar formation and its expression is inversely related to the periods of vascularization and alveolarization in the developing lung. Hence the study of EMAP II could play a vital role in studying and devising appropriate therapeutics for diseases of aberrant lung development, such as BPD. Herein, we review the vascular contribution to lung development and the implications that vascular mediators such as EMAP II have in distal lung formation during the vulnerable stage of alveolar genesis

Augmentation of Nab-Paclitaxel Chemotherapy Response by Mechanistically Diverse Antiangiogenic Agents in Preclinical Gastric Cancer Models

Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly recommended as a fundamental treatment for metastatic GAC; however, standard treatment has not been established yet. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated therapeutic benefits of mechanistically diverse antiangiogenic agents in combination with nab-paclitaxel, a next-generation taxane, in preclinical models of GAC. Murine survival studies were performed in peritoneal dissemination models, whereas tumor growth studies were performed in subcutaneous GAC cell-derived or patient-derived xenografts. The mechanistic evaluation involved IHC and Immunoblot analysis in tumor samples. Nab-paclitaxel increased animal survival that was further improved by the addition of antiangiogenic agents ramucirumab (or its murine version DC101), cabozantinib and nintedanib. Nab-paclitaxel combination with nintedanib was most effective in improving animal survival, always greater than 300% over control. In cell-derived subcutaneous xenografts, nab-paclitaxel reduced tumor growth while all three antiangiogenic agents enhanced this effect, with nintedanib demonstrating the greatest inhibition. Furthermore, in GAC patient-derived xenografts the combination of nab-paclitaxel and nintedanib reduced tumor growth over single agents alone. Tumor tissue analysis revealed that ramucirumab and cabozantinib only reduced tumor vasculature, whereas nintedanib in addition significantly reduced tumor cell proliferation and increased apoptosis. Effects of nab-paclitaxel, a promising chemotherapeutic agent for GAC, can be enhanced by new-generation antiangiogenic agents, especially nintedanib. The data suggest that nab-paclitaxel combinations with multitargeted antiangiogenic agents carry promising potential for improving clinical GAC therapy

Inhibition of the MEK/ERK pathway augments nab-paclitaxel-based chemotherapy effects in preclinical models of pancreatic cancer

Nab-paclitaxel (NPT) combination with gemcitabine (Gem) represents the standard chemotherapy for pancreatic ductal adenocarcinoma (PDAC). Genetic alterations of the RAS/RAF/MEK/ERK (MAPK) signaling pathway yielding constitutive activation of the ERK cascade have been implicated as drivers of PDAC. Inhibition of downstream targets in the RAS-MAPK cascade such as MEK remains a promising therapeutic strategy. The efficacy of trametinib (Tra), a small molecule inhibitor of MEK1/2 kinase activity, in combination with nab-paclitaxel-based chemotherapy was evaluated in preclinical models of PDAC. The addition of trametinib to chemotherapy regimens showed a trend for an additive effect on tumor growth inhibition in subcutaneous AsPC-1 and Panc-1 PDAC xenografts. In a peritoneal dissemination model, median animal survival compared to controls (20 days) was increased after therapy with NPT (33 days, a 65% increase), Tra (31 days, a 55% increase), NPT+Tra (37 days, a 85% increase), NPT+Gem (39 days, a 95% increase) and NPT+Gem+Tra (49 days, a 145% increase). Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition. Trametinib effects were specifically accompanied by a decrease in phospho-ERK and an increase in cleaved caspase-3 and cleaved PARP-1 proteins. These findings suggest that the effects of nab-paclitaxel-based chemotherapy can be enhanced through specific inhibition of MEK1/2 kinase activity, and supports the clinical application of trametinib in combination with standard nab-paclitaxel-based chemotherapy in PDAC patients

N-terminus of pro-EMAP II regulates its binding with C-terminus, Arginyl-tRNA Synthetase, and Neurofilament light protein

Pro-EMAP II, one component of the Multi-Aminoacyl tRNA Synthetase (MSC) Complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development and tumor growth. Recent studies determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of pro-EMAP IIs C-terminus was reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that pro-EMAP IIs N-terminus had an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine-zipper in the N-terminus of human pro-EMAP II protein (amino acid residues 1-70), which can form specific strip-like punctate structures. Through GFP punctate analysis, we uncovered that pro-EMAP IIs C-terminus (147-312 amino acid residues) can repress the GFP punctate formation. Pull-down assays confirmed the binding between pro-EMAP II N-terminus and its C-terminus is mediated by a putative leucine-zipper. Furthermore, the pro-EMAP II 1-70 aa region was identified as the binding partner of the arginyl-tRNA synthetase (RARS), a polypeptide of MSC complex. We also determined that the punctate GFP pro-EMAP II 1-70aa aggregate co-localizes and binds to the neurofilament light (NFL) subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of Pro-EMAP II protein and suggest a role of this protein in the pathological neurodegenerative diseases

α5β1 integrin mediates pulmonary epithelial cyst formation

BACKGROUND: Formation of the epithelial cyst involves the establishment of apical-basolateral polarity through a series of cellular interactions that are in part mediated by the extracellular matrix (ECM). We report that in a three-dimensional multi-cellular self-assembly model of lung development, α5 integrin regulates epithelial cyst formation through organization of soluble fibronectin matrix into insoluble fibrils through a process called fibrillogenesis. RESULTS: Dissociated murine embryonic lung cells self-assemble into three-dimensional pulmonary bodies that are dependent on α5β1 integrin mediated fibrillogenesis for cell-cell mediated self-assembly: compaction and epithelial cyst formation. Knockdown of α5 integrin resulted in a significant increase in another mediator of fibrillogenesis, αV integrin. Compensatory increased expression of another mediator of fibrillogenesis, αV integrin, was not sufficient to normalize epithelial cyst formation. Loss of α5 integrin-mediated fibrillogenesis perturbed the ability of clustered epithelial cells to establish clear polarity, loss of epithelial cell pyramidal shape, and disrupted apical F-actin-rich deposition. Lack of rich central epithelial localization of F-actin cytoskeleton and Podocalyxin suggests that loss of α5 integrin-mediated fibrillogenesis interferes with the normal cytoskeleton organization that facilitates epithelial cysts polarization. CONCLUSIONS: We conclude that lung epithelial cyst formation in development is mediated in part by α5β1 integrin dependent fibrillogenesi

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 antiangiogenic 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 diseas

Smac mimetic-derived augmentation of chemotherapeutic response in experimental pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to conventional chemotherapy, in part due to the overexpression of inhibitors of apoptosis proteins (IAPs). Smac is an endogenous IAP-antagonist, which renders synthetic Smac mimetics attractive anticancer agents. We evaluated the benefits of combining a Smac mimetic, JP1201 (JP), with conventional chemotherapy agents used for PDAC management.</p> <p>Methods</p> <p>Cell viability assays and protein expression analysis were performed using WST-1 reagent and Western blotting, respectively. Apoptosis was detected by annexin V/propidium iodide staining. <it>In vivo </it>tumor growth and survival studies were performed in murine PDAC xenografts.</p> <p>Results</p> <p>JP and gemcitabine (Gem) inhibited PDAC cell proliferation with additive effects in combination. The percentage of early apoptotic cells in controls, JP, Gem and JP + Gem was 17%, 26%, 26% and 38%, respectively. JP-induced apoptosis was accompanied by PARP-1 cleavage. Similar additive anti-proliferative effects were seen for combinations of JP with doxorubicin (Dox) and docetaxel (DT). The JP + Gem combination caused a 30% decrease in tumor size <it>in vivo </it>compared to controls. Median animal survival was improved significantly in mice treated with JP + Gem (38 d) compared to controls (22 d), JP (28 d) or Gem (32 d) (p = 0.01). Animal survival was also improved with JP + DT treatment (32 d) compared to controls (16 d), JP (21 d) or DT alone (27 d).</p> <p>Conclusions</p> <p>These results warrant further exploration of strategies that promote chemotherapy-induced apoptosis of tumors and highlight the potential of Smac mimetics in clinical PDAC therapy.</p

Augmentation of response to nab-paclitaxel by inhibition of insulin-like growth factor (IGF) signaling in preclinical pancreatic cancer models

Nab-paclitaxel has recently shown greater efficacy in pancreatic ductal adenocarcinoma (PDAC). Insulin like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC and correlate with aggressive tumor phenotype and poor prognosis. We evaluated the improvement in nab-paclitaxel response by addition of BMS-754807, a small molecule inhibitor of IGF-1R/IR signaling, in preclinical PDAC models. In subcutaneous xenografts using AsPC-1 cells, average net tumor growth in different therapy groups was 248.3 mm3 in controls, 42.4 mm3 after nab-paclitaxel (p = 0.002), 93.3 mm3 after BMS-754807 (p = 0.01) and 1.9 mm3 after nab-paclitaxel plus BMS-754807 (p = 0.0002). In subcutaneous xenografts using Panc-1 cells, average net tumor growth in different therapy groups was: 294.3 mm3 in controls, 23.1 mm3 after nab-paclitaxel (p = 0.002), 118.2 mm3 after BMS-754807 (p = 0.02) and -87.4 mm3 (tumor regression) after nab-paclitaxel plus BMS-754807 (p = 0.0001). In peritoneal dissemination model using AsPC-1 cells, median animal survival was increased compared to controls (21 days) after therapy with nab-paclitaxel (40 days, a 90% increase, p = 0.002), BMS-754807 (27 days, a 29% increase, p = 0.01) and nab-paclitaxel plus BMS-754807 (47 days, a 124% increase, p = 0.005), respectively. Decrease in proliferation and increase in apoptosis by nab-paclitaxel and BMS-754807 therapy correlated with their in vivo antitumor activity. In vitro analysis revealed that the addition of IC25 dose of BMS-754807 decreased the nab-paclitaxel IC50 of PDAC cell lines. BMS-754807 therapy decreased phospho-IGF-1R/IR and phospho-AKT expression, and increased cleavage of caspase-3 and PARP-1. These results support the potential of BMS-754807 in combination with nab-paclitaxel as an effective targeting option for pancreatic cancer therapy