Publications by authors named "Giuseppe Fiume"

44 Publications

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy.

Viruses 2021 04 9;13(4). Epub 2021 Apr 9.

Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.
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http://dx.doi.org/10.3390/v13040649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070105PMC
April 2021

Exacerbation of Allergic Airway Inflammation in Mice Lacking ECTO-5'-Nucleotidase (CD73).

Front Pharmacol 2020 30;11:589343. Epub 2020 Nov 30.

Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy.

The airways are a target tissue of type I allergies and atopy is the main etiological factor of bronchial asthma. A predisposition to allergy and individual response to allergens are dependent upon environmental and host factors. Early studies performed to clarify the role of extracellular adenosine in the airways highlighted the importance of adenosine-generating enzymes CD73, together with CD39, as an innate protection system against lung injury. In experimental animals, deletion of CD73 has been associated with immune and autoimmune diseases. Our experiments have been performed to investigate the role of CD73 in the assessment of allergic airway inflammation following sensitization. We found that in CD73 mice sensitization, induced by subcutaneous ovalbumin (OVA) administration, increased signs of airway inflammation and atopy developed, characterized by high IgE plasma levels and increased pulmonary cytokines, reduced frequency of lung CD4CD25+Foxp3+ T cells, but without bronchial hyperreactivity, compared to sensitized wild type mice. Our results provide evidence that the lack of CD73 causes an uncontrolled allergic sensitization, suggesting that CD73 is a key molecule at the interface between innate and adaptive immune response. The knowledge of host immune factors controlling allergic sensitization is of crucial importance and might help to find preventive interventions that could act before an allergy develops.
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http://dx.doi.org/10.3389/fphar.2020.589343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734328PMC
November 2020

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import.

Nat Commun 2020 12 1;11(1):6145. Epub 2020 Dec 1.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.
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http://dx.doi.org/10.1038/s41467-020-19871-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708531PMC
December 2020

Role of Chronic Lymphocytic Leukemia (CLL)-Derived Exosomes in Tumor Progression and Survival.

Pharmaceuticals (Basel) 2020 Sep 14;13(9). Epub 2020 Sep 14.

Department of Experimental and Clinical Medicine - University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.

Chronic lymphocytic leukemia (CLL) is a B-lymphoproliferative disease, which consists of the abnormal proliferation of CD19/CD5/CD20/CD23 positive lymphocytes in blood and lymphoid organs, such as bone marrow, lymph nodes and spleen. The neoplastic transformation and expansion of tumor B cells are commonly recognized as antigen-driven processes, mediated by the interaction of antigens with the B cell receptor (BCR) expressed on the surface of B-lymphocytes. The survival and progression of CLL cells largely depend on the direct interaction of CLL cells with receptors of accessory cells of tumor microenvironment. Recently, much interest has been focused on the role of tumor release of small extracellular vesicles (EVs), named exosomes, which incorporate a wide range of biologically active molecules, particularly microRNAs and proteins, which sustain the tumor growth. Here, we will review the role of CLL-derived exosomes as diagnostic and prognostic biomarkers of the disease.
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http://dx.doi.org/10.3390/ph13090244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557731PMC
September 2020

Uncovering the Exosomes Diversity: A Window of Opportunity for Tumor Progression Monitoring.

Pharmaceuticals (Basel) 2020 Aug 4;13(8). Epub 2020 Aug 4.

Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, 88100 Catanzaro, Italy.

Cells can communicate through special "messages in the bottle", which are recorded in the bloodstream inside vesicles, namely exosomes. The exosomes are nanovesicles of 30-100 nm in diameter that carry functionally active biological material, such as proteins, messanger RNA (mRNAs), and micro RNA (miRNAs). Therefore, they are able to transfer specific signals from a parental cell of origin to the surrounding cells in the microenvironment and to distant organs through the circulatory and lymphatic stream. More and more interest is rising for the pathological role of exosomes produced by cancer cells and for their potential use in tumor monitoring and patient follow up. In particular, the exosomes could be an appropriate index of proliferation and cancer cell communication for monitoring the minimal residual disease, which cannot be easily detectable by common diagnostic and monitoring techniques. The lack of unequivocal markers for tumor-derived exosomes calls for new strategies for exosomes profile characterization aimed at the adoption of exosomes as an official tumor biomarker for tumor progression monitoring.
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http://dx.doi.org/10.3390/ph13080180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464894PMC
August 2020

Influence of Fibroblasts on Mammary Gland Development, Breast Cancer Microenvironment Remodeling, and Cancer Cell Dissemination.

Cancers (Basel) 2020 Jun 26;12(6). Epub 2020 Jun 26.

Department of Public Health, University of Naples Federico II, 80131 Naples, Italy.

The stromal microenvironment regulates mammary gland development and tumorigenesis. In normal mammary glands, the stromal microenvironment encompasses the ducts and contains fibroblasts, the main regulators of branching morphogenesis. Understanding the way fibroblast signaling pathways regulate mammary gland development may offer insights into the mechanisms of breast cancer (BC) biology. In fact, the unregulated mammary fibroblast signaling pathways, associated with alterations in extracellular matrix (ECM) remodeling and branching morphogenesis, drive breast cancer microenvironment (BCM) remodeling and cancer growth. The BCM comprises a very heterogeneous tissue containing non-cancer stromal cells, namely, breast cancer-associated fibroblasts (BCAFs), which represent most of the tumor mass. Moreover, the different components of the BCM highly interact with cancer cells, thereby generating a tightly intertwined network. In particular, BC cells activate recruited normal fibroblasts in BCAFs, which, in turn, promote BCM remodeling and metastasis. Thus, comparing the roles of normal fibroblasts and BCAFs in the physiological and metastatic processes, could provide a deeper understanding of the signaling pathways regulating BC dissemination. Here, we review the latest literature describing the structure of the mammary gland and the BCM and summarize the influence of epithelial-mesenchymal transition (EpMT) and autophagy in BC dissemination. Finally, we discuss the roles of fibroblasts and BCAFs in mammary gland development and BCM remodeling, respectively.
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http://dx.doi.org/10.3390/cancers12061697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352995PMC
June 2020

Insights about MYC and Apoptosis in B-Lymphomagenesis: An Update from Murine Models.

Int J Mol Sci 2020 Jun 15;21(12). Epub 2020 Jun 15.

Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.

The balance between cell survival and cell death represents an essential part of human tissue homeostasis, while altered apoptosis contributes to several pathologies and can affect the treatment efficacy. Impaired apoptosis is one of the main cancer hallmarks and some types of lymphomas harbor mutations that directly affect key regulators of cell death (such as BCL-2 family members). The development of novel techniques in the field of immunology and new animal models has greatly accelerated our understanding of oncogenic mechanisms in MYC-associated lymphomas. Mouse models are a powerful tool to reveal multiple genes implicated in the genesis of lymphoma and are extensively used to clarify the molecular mechanism of lymphoma, validating the gene function. Key features of MYC-induced apoptosis will be discussed here along with more recent studies on MYC direct and indirect interactors, including their cooperative action in lymphomagenesis. We review our current knowledge about the role of MYC-induced apoptosis in B-cell malignancies, discussing the transcriptional regulation network of MYC and regulatory feedback action of miRs during MYC-driven lymphomagenesis. More importantly, the finding of new modulators of apoptosis now enabling researchers to translate the discoveries that have been made in the laboratory into clinical practice to positively impact human health.
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http://dx.doi.org/10.3390/ijms21124265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352788PMC
June 2020

Metabolic Plasticity of Melanoma Cells and Their Crosstalk With Tumor Microenvironment.

Front Oncol 2020 22;10:722. Epub 2020 May 22.

Department of Public Health, University of Naples Federico II, Naples, Italy.

Cutaneous melanoma (CM) is a highly aggressive and drug resistant solid tumor, showing an impressive metabolic plasticity modulated by oncogenic activation. In particular, melanoma cells can generate adenosine triphosphate (ATP) during cancer progression by both cytosolic and mitochondrial compartments, although CM energetic request mostly relies on glycolysis. The upregulation of glycolysis is associated with constitutive activation of BRAF/MAPK signaling sustained by BRAF kinase mutant. In this scenario, the growth and progression of CM are strongly affected by melanoma metabolic changes and interplay with tumor microenvironment (TME) that sustain tumor development and immune escape. Furthermore, CM metabolic plasticity can induce a metabolic adaptive response to BRAF/MEK inhibitors (BRAFi/MEKi), associated with the shift from glycolysis toward oxidative phosphorylation (OXPHOS). Therefore, in this review article we survey the metabolic alterations and plasticity of CM, its crosstalk with TME that regulates melanoma progression, drug resistance and immunosurveillance. Finally, we describe hallmarks of melanoma therapeutic strategies targeting the shift from glycolysis toward OXPHOS.
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http://dx.doi.org/10.3389/fonc.2020.00722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256186PMC
May 2020

Detection of chronic lymphocytic leukemia subpopulations in peripheral blood by phage ligands of tumor immunoglobulin B cell receptors.

Leukemia 2021 02 1;35(2):610-614. Epub 2020 Jun 1.

Department of Experimental and Clinical Medicine, "Magna Græcia" University of Catanzaro, Campus "S. Venuta", Viale Europa, Germaneto, 88100, Catanzaro, Italy.

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http://dx.doi.org/10.1038/s41375-020-0885-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862058PMC
February 2021

Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy.

Int J Mol Sci 2020 Mar 30;21(7). Epub 2020 Mar 30.

Department of Experimental and Clinical Medicine, University "Magna Græcia" of Catanzaro, 88100 Catanzaro, Italy.

The T cells are key players of the response to checkpoint blockade immunotherapy (CBI) and monitoring the strength and specificity of antitumor T-cell reactivity remains a crucial but elusive component of precision immunotherapy. The entire assembly of T-cell receptor (TCR) sequences accounts for antigen specificity and strength of the T-cell immune response. The TCR repertoire hence represents a "footprint" of the conditions faced by T cells that dynamically evolves according to the challenges that arise for the immune system, such as tumor neo-antigenic load. Hence, TCR repertoire analysis is becoming increasingly important to comprehensively understand the nature of a successful antitumor T-cell response, and to improve the success and safety of current CBI.
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http://dx.doi.org/10.3390/ijms21072378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177412PMC
March 2020

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.

Nat Chem Biol 2020 04 9;16(4):469-478. Epub 2020 Mar 9.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
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http://dx.doi.org/10.1038/s41589-020-0483-3DOI Listing
April 2020

IBTK Haploinsufficiency Affects the Tumor Microenvironment of Myc-Driven Lymphoma in E-myc Mice.

Int J Mol Sci 2020 Jan 30;21(3). Epub 2020 Jan 30.

Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.

The tumor microenvironment is a dynamic and interactive supporting network of various components, including blood vessels, cytokines, chemokines, and immune cells, which sustain the tumor cell's survival and growth. Murine models of lymphoma are useful to study tumor biology, the microenvironment, and mechanisms of response to therapy. Lymphomas are heterogeneous hematologic malignancies, and the complex microenvironment from which they arise and their multifaceted genetic basis represents a challenge for the generation and use of an appropriate murine model. So, it is important to choose the correct methodology. Recently, we supported the first evidence on the pro-oncogenic action of IBTK in Myc-driven B cell lymphomagenesis in mice, inhibiting apoptosis in the pre-cancerous stage. We used the transgenic mouse model of non-Hodgkin's lymphoma and hemizygous mice to evaluate the tumor development of Myc-driven lymphoma. Here, we report that the allelic loss of alters the immunophenotype of Myc-driven B cell lymphomas, increasing the rate of pre-B cells and affecting the tumor microenvironment in mice. In particular, we observed enhanced tumor angiogenesis, increasing pro-angiogenic and lymphangiogenic factors, such as VEGF, MMP-9, CCL2, and VEGFD, and a significant recruitment of tumor-associated macrophages in lymphomas of compared to mice. In summary, these results indicate that haploinsufficiency promotes Myc tumor development by modifying the tumor microenvironment.
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http://dx.doi.org/10.3390/ijms21030885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038122PMC
January 2020

Development of a Stromal Microenvironment Experimental Model Containing Proto-Myofibroblast Like Cells and Analysis of Its Crosstalk with Melanoma Cells: A New Tool to Potentiate and Stabilize Tumor Suppressor Phenotype of Dermal Myofibroblasts.

Cells 2019 11 14;8(11). Epub 2019 Nov 14.

Department of Public Health, University of Naples Federico II, 80131 Naples, Italy.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.
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http://dx.doi.org/10.3390/cells8111435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912587PMC
November 2019

Molecular modelling of epitopes recognized by neoplastic B lymphocytes in Chronic Lymphocytic Leukemia.

Eur J Med Chem 2020 Jan 1;185:111838. Epub 2019 Nov 1.

Department of Health Sciences, University "Magna Græcia" of Catanzaro, Campus "S. Venuta", Viale Europa, Germaneto, 88100, Catanzaro, Italy; Net4Science srl, University "Magna Græcia", Campus Salvatore Venuta, Viale Europa, Catanzaro, 88100, Italy. Electronic address:

Identification of epitopes recognized by tumour B cells could provide insights into the molecular mechanisms of B cell tumorigenesis through aberrant B cell receptor (BCR) signalling. Here, we analysed the structure of eleven peptides binders of BCRs expressed in Chronic Lymphocytic Leukemia (CLL) patients in order to identify the chemical features required for cross-reactive binding to different CLL clonotypes. Four cross-reactive (CR) and seven no-cross-reactive (NCR) peptides were analysed by means of GRID molecular interaction fields, ligand-based pharmacophore and 3D-QSAR approaches. Based on pharmacophore model, two peptides were generated by specific amino acids substitutions of the parental NCR peptides; these new peptides resumed the common chemical features of CR peptides and bound the CLL BCR clonotypes recognized by CR peptides and parental NCR peptides. Thus, our computational approach guided the pharmacophore modelling of CR peptides. In perspective, peptide binders of CLL BCR clonotypes could represent a powerful tool for computational modelling of epitopes recognized by tumour B cells clones.
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http://dx.doi.org/10.1016/j.ejmech.2019.111838DOI Listing
January 2020

Insights into Thymus Development and Viral Thymic Infections.

Viruses 2019 09 9;11(9). Epub 2019 Sep 9.

Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, 88100 Catanzaro, Italy.

T-cell development in the thymus is a complex and highly regulated process, involving a wide variety of cells and molecules which orchestrate thymocyte maturation into either CD4 or CD8 single-positive (SP) T cells. Here, we briefly review the process regulating T-cell differentiation, which includes the latest advances in this field. In particular, we highlight how, starting from a pool of hematopoietic stem cells in the bone marrow, the sequential action of transcriptional factors and cytokines dictates the proliferation, restriction of lineage potential, T-cell antigen receptors (TCR) gene rearrangements, and selection events on the T-cell progenitors, ultimately leading to the generation of mature T cells. Moreover, this review discusses paradigmatic examples of viral infections affecting the thymus that, by inducing functional changes within this lymphoid gland, consequently influence the behavior of peripheral mature T-lymphocytes.
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http://dx.doi.org/10.3390/v11090836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784209PMC
September 2019

Discovery of carboxylic acid reductase (CAR) from Thermothelomyces thermophila and its evaluation for vanillin synthesis.

J Biotechnol 2019 Oct 13;304:44-51. Epub 2019 Aug 13.

Austrian Center of Industrial Biotechnology, Petersgasse 14, Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, Graz, Austria. Electronic address:

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (T) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L of vanillin. The space time yield of 0.029 g L h indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.
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http://dx.doi.org/10.1016/j.jbiotec.2019.08.007DOI Listing
October 2019

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection.

Sci Rep 2019 07 18;9(1):10471. Epub 2019 Jul 18.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090, Vienna, Austria.

Host factor requirements for different classes of viruses have not been fully unraveled. Replication of the viral genome and synthesis of viral proteins within the human host cell are associated with an increased demand for nutrients and specific metabolites. With more than 400 acknowledged members to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cellular response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that the loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes suggests a role for both of these transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.
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http://dx.doi.org/10.1038/s41598-019-46952-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639343PMC
July 2019

IBTK contributes to B-cell lymphomagenesis in Eμ-myc transgenic mice conferring resistance to apoptosis.

Cell Death Dis 2019 04 11;10(4):320. Epub 2019 Apr 11.

Department of Experimental and Clinical Medicine, University Magna Græcia of Catanzaro, Catanzaro, 88100, Italy.

Increasing evidence supports the involvement of IBTK in cell survival and tumor growth. Previously, we have shown that IBTK RNA interference affects the wide genome expression and RNA splicing in cell-type specific manner. Further, the expression of IBTK gene progressively increases from indolent to aggressive stage of chronic lymphocytic leukemia and decreases in disease remission after therapy. However, the role of IBTK in tumorigenesis has not been elucidated. Here, we report that loss of the murine Ibtk gene raises survival and delays tumor onset in Eμ-myc transgenic mice, a preclinical model of Myc-driven lymphoma. In particular, we found that the number of pre-cancerous B cells of bone marrow and spleen is reduced in IbtkEμ-myc mice owing to impaired viability and increased apoptosis, as measured by Annexin V binding, Caspase 3/7 cleavage assays and cell cycle profile analysis. Instead, the proliferation rate of pre-cancerous B cells is unaffected by the loss of Ibtk. We observed a direct correlation between Ibtk and myc expression and demonstrated a Myc-dependent regulation of Ibtk expression in murine B cells, human hematopoietic and nonhematopoietic cell lines by analysis of ChIP-seq data. By tet-repressible Myc system, we confirmed a Myc-dependent expression of IBTK in human B cells. Further, we showed that Ibtk loss affected the main apoptotic pathways dependent on Myc overexpression in pre-cancerous Eμ-myc mice, in particular, MCL-1 and p53. Of note, we found that loss of IBTK impaired cell cycle and increased apoptosis also in a human epithelial cell line, HeLa cells, in Myc-independent manner. Taken together, these results suggest that Ibtk sustains the oncogenic activity of Myc by inhibiting apoptosis of murine pre-cancerous B cells, as a cell-specific mechanism. Our findings could be relevant for the development of IBTK inhibitors sensitizing tumor cells to apoptosis.
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http://dx.doi.org/10.1038/s41419-019-1557-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459904PMC
April 2019

Activation of NF-κB in B cell receptor signaling through Bruton's tyrosine kinase-dependent phosphorylation of IκB-α.

J Mol Med (Berl) 2019 05 19;97(5):675-690. Epub 2019 Mar 19.

Department of Clinical and Experimental Medicine, University of Catanzaro "Magna Græcia", Viale Europa, Germaneto, 88100, Catanzaro, Italy.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5 min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30 s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.
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http://dx.doi.org/10.1007/s00109-019-01777-xDOI Listing
May 2019

Revisiting Bacterial Ubiquitin Ligase Effectors: Weapons for Host Exploitation.

Int J Mol Sci 2018 Nov 13;19(11). Epub 2018 Nov 13.

Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, 88100 Catanzaro, Italy.

Protein ubiquitylation plays a central role in eukaryotic cell physiology. It is involved in several regulatory processes, ranging from protein folding or degradation, subcellular localization of proteins, vesicular trafficking and endocytosis to DNA repair, cell cycle, innate immunity, autophagy, and apoptosis. As such, it is reasonable that pathogens have developed a way to exploit such a crucial system to enhance their virulence against the host. Hence, bacteria have evolved a wide range of effectors capable of mimicking the main players of the eukaryotic ubiquitin system, in particular ubiquitin ligases, by interfering with host physiology. Here, we give an overview of this topic and, in particular, we detail and discuss the mechanisms developed by pathogenic bacteria to hijack the host ubiquitination system for their own benefit.
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http://dx.doi.org/10.3390/ijms19113576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274744PMC
November 2018

The expression of inhibitor of bruton's tyrosine kinase gene is progressively up regulated in the clinical course of chronic lymphocytic leukaemia conferring resistance to apoptosis.

Cell Death Dis 2018 01 9;9(1):13. Epub 2018 Jan 9.

Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.

Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy with a variable clinical outcome. Biomarkers of CLL progression are required for optimising prognosis and therapy. The Inhibitor of Bruton's tyrosine kinase-isoform α (IBTKα) gene encodes a substrate receptor of Cullin 3-dependent E3 ubiquitin ligase, and promotes cell survival in response to the reticulum stress. Searching for novel markers of CLL progression, we analysed the expression of IBTKα in the peripheral blood B-cells of CLL patients, before and after first line therapy causing remission. The expression of IBTKα was significantly increased in disease progression, and decreased in remission after chemotherapy. Consistently with a pro-survival action, RNA interference of IBTKα increased the spontaneous and Fludarabine-induced apoptosis of MEC-1 CLL cells, and impaired the cell cycle of DeFew B-lymphoma cells by promoting the arrest in G0/G1 phase and apoptosis. Consistently, RNA interference of IBTKα up regulated the expression of pro-apoptotic genes, including TNF, CRADD, CASP7, BNIP3 and BIRC3. Our results indicate that IBTKα is a novel marker of CLL progression promoting cell growth and resistance to apoptosis. In this view, IBTKα may represent an attractive cancer drug target for counteracting the therapy-resistance of tumour cells.
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http://dx.doi.org/10.1038/s41419-017-0026-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849039PMC
January 2018

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

Mol Cancer 2017 10 13;16(1):159. Epub 2017 Oct 13.

Department of Experimental and Clinical Medicine, University of Catanzaro "Magna Graecia,", Catanzaro, Italy.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.
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http://dx.doi.org/10.1186/s12943-017-0730-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640902PMC
October 2017

Generation and analysis of spheroids from human primary skin myofibroblasts: an experimental system to study myofibroblasts deactivation.

Cell Death Discov 2017 17;3:17038. Epub 2017 Jul 17.

Department of Public Health, University of Naples Federico II, Naples 80131, Italy.

Myofibroblasts are activated fibroblasts involved in tissue repair and cancer. They are characterized by expression of -smooth muscle actin (-SMA), immunoregulatory phenotype and paracrine interaction with normal and tumorigenic cells leading to cell proliferation. At the end of wound-healing myofibroblasts undergo apoptotic cell death, whereas -activated fibroblasts are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response. Furthermore, myofibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this study, we generated and analysed clusters such as spheroids from human primary cutaneous myofibroblasts, which represent a part of stromal microenvironment better than established cell lines. Therefore, we evaluated apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation did not trigger apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of -SMA in protein extracts of spheroids, the cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts have undergone a deactivation process within spheroids. The cells of spheroids reverted to adhesion growth preserved their proliferation capability and can re-acquire a myofibroblastic phenotype. Moreover, the spontaneous formation of clusters on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and suggests that fibroblast clusters could be a cell reservoir regulating tissues turnover.
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http://dx.doi.org/10.1038/cddiscovery.2017.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511858PMC
July 2017

Selective Enzymatic Transformation to Aldehydes by Fungal Carboxylate Reductase from .

Adv Synth Catal 2016 11 4;358(21):3414-3421. Epub 2016 Oct 4.

acib GmbH Petersgasse 14 8010 Graz Austria; Institute of Molecular Biotechnology Graz University of Technology NAWI Graz Petersgasse 14 8010 Graz Austria.

The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate-reducing enzymes (CARs) with the sequence of a fungal CAR from OR74A (CAR). CAR was efficiently expressed in using an autoinduction protocol at low temperature. It was purified and characterized , revealing a broad substrate acceptance, a pH optimum at pH 5.5-6.0, a of 45 °C and inhibition by the co-product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of CAR was demonstrated in a whole-cell biotransformation using the K-12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.
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http://dx.doi.org/10.1002/adsc.201600914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129534PMC
November 2016

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

Int J Mol Sci 2016 Nov 7;17(11). Epub 2016 Nov 7.

Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy.

The gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.
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http://dx.doi.org/10.3390/ijms17111848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133848PMC
November 2016

The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

Sci Rep 2016 05 11;6:25879. Epub 2016 May 11.

Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.

Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.
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http://dx.doi.org/10.1038/srep25879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863168PMC
May 2016

Single-cell real-time imaging of transgene expression upon lipofection.

Biochem Biophys Res Commun 2016 05 21;474(1):8-14. Epub 2016 Mar 21.

Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy. Electronic address:

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of "symmetry" in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents.
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http://dx.doi.org/10.1016/j.bbrc.2016.03.088DOI Listing
May 2016

Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice.

Sci Rep 2015 Sep 7;5:13864. Epub 2015 Sep 7.

Department of Experimental and Clinical Medicine, University of Catanzaro "Magna Graecia", Viale Europa, 88100, Catanzaro, Italy.

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4(+) and CD8(+) T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.
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http://dx.doi.org/10.1038/srep13864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561375PMC
September 2015

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy.

Cancer Cell Int 2015 7;15:50. Epub 2015 May 7.

Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, I-80131 Naples, Italy ; Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini, I-80131 Naples, Italy.

Background: Despite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents.

Methods: Here, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively.

Results: Compared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes.

Conclusions: Peptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.
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http://dx.doi.org/10.1186/s12935-015-0202-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433068PMC
May 2015

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation.

J Biol Chem 2015 May 16;290(22):13958-71. Epub 2015 Apr 16.

From the Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, 88100 Catanzaro, Italy

The human inhibitor of Bruton's tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3(IBTK) complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3(IBTK) for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3(IBTK) regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5'-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3(IBTK) as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.
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http://dx.doi.org/10.1074/jbc.M114.634535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447969PMC
May 2015