148 results on '"Provenzani, Alessandro"'
Search Results
102. Glucosylation of Isatin-3-Oxime followed by 2Din situNMR in Plant Cells at Highest Magnetic Field without Labelling
- Author
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Hinse, Christiane, primary, Unger, Matthias, additional, Provenzani, Alessandro, additional, and Stöckigt, Joachim, additional
- Published
- 2001
- Full Text
- View/download PDF
103. Intramolecular Hetero Diels−Alder Reactions of α,α’-Dioxosulfines − A New Access to the [3.3.1]-Bicyclic Skeleton
- Author
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Capozzi, Giuseppe, primary, Menichetti, Stefano, additional, Nativi, Cristina, additional, and Provenzani, Alessandro, additional
- Published
- 2000
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104. Targeting the Multifaceted HuR Protein, Benefits and Caveats
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Zucal, Chiara, D’Agostino, Vito, Loffredo, Rosa, Mantelli, Barbara, Thongon, Natthakan, Lal, Preet, Latorre, Elisa, and Provenzani, Alessandro
- Abstract
The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3’UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR’s post-transcriptional control and the efforts made to identify specific HuR inhibitors.
- Published
- 2015
105. PER2 promotes glucose storage to liver glycogen during feeding and acute fasting by inducing Gys2 PTG and G L expression.
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Zani, Fabio, Breasson, Ludovic, Becattini, Barbara, Vukolic, Ana, Montani, Jean-Pierre, Albrecht, Urs, Provenzani, Alessandro, Ripperger, Juergen A., and Solinas, Giovanni
- Abstract
Abstract: The interplay between hepatic glycogen metabolism and blood glucose levels is a paradigm of the rhythmic nature of metabolic homeostasis. Here we show that mice lacking a functional PER2 protein (Per2
Brdm1 ) display reduced fasting glycemia, altered rhythms of hepatic glycogen accumulation, and altered rhythms of food intake. Per2Brdm1 mice show reduced hepatic glycogen content and altered circadian expression during controlled fasting and refeeding. Livers from Per2Brdm1 mice display reduced glycogen synthase protein levels during refeeding, and increased glycogen phosphorylase activity during fasting. The latter is explained by PER2 action on the expression of the adapter proteins PTG and GL , which target the protein phosphatase-1 to glycogen to decrease glycogen phosphorylase activity. Finally, PER2 interacts with genomic regions of Gys2, PTG, and GL . These results indicate an important role for PER2 in the hepatic transcriptional response to feeding and acute fasting that promotes glucose storage to liver glycogen. [Copyright &y& Elsevier]- Published
- 2013
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106. Life and death of the mRNA in the cytoplasm.
- Author
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Denti, Michela A., Viero, Gabriella, Provenzani, Alessandro, Quattrone, Alessandro, and Macchi, Paolo
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- 2013
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107. Browsing gene banks for Fe2S2 ferredoxins and structural modeling of 88 plant-type sequences: An analysis of fold and function.
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Bertini, Ivano, Luchinat, Claudio, Provenzani, Alessandro, Rosato, Antonio, and Vasos, Paul R.
- Published
- 2002
- Full Text
- View/download PDF
108. Glucosylation of Isatin-3-Oxime followed by 2D in situ NMR in Plant Cells at Highest Magnetic Field without Labelling.
- Author
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Hinse, Christiane, Unger, Matthias, Provenzani, Alessandro, and Stöckigt, Joachim
- Published
- 2001
- Full Text
- View/download PDF
109. Browsing gene banks for Fe2S2ferredoxins and structural modeling of 88 plant‐type sequences: An analysis of fold and function
- Author
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Bertini, Ivano, Luchinat, Claudio, Provenzani, Alessandro, Rosato, Antonio, and Vasos, Paul R.
- Abstract
One‐hundred‐and‐seventy‐nine sequences of Fe2S2ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster‐binding patterns, these sequences were divided into three groups: those containing the CX4CX2CXnC pattern (plant‐type ferredoxins), those with the CX5CX2CXnC pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster‐binding patterns: 88 sequences had the CX4CX2CX29C pattern, and 29 had the CX4CX2CXmC (m≠29) pattern. The structures of the 88 ferredoxins with the CX4CX2CX29C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 Å of the backbone root‐mean‐square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three‐dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent‐accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of protein—protein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4CX2CXmC (m≠29) cluster‐binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup. Proteins 2002;46:110–127. © 2001 Wiley‐Liss, Inc.
- Published
- 2002
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- View/download PDF
110. Browsing gene banks for Fe<INF>2</INF>S<INF>2</INF> ferredoxins and structural modeling of 88 plant-type sequences: An analysis of fold and function
- Author
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Bertini, Ivano, Luchinat, Claudio, Provenzani, Alessandro, Rosato, Antonio, and Vasos, Paul R.
- Abstract
One-hundred-and-seventy-nine sequences of Fe
2 S2 ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster-binding patterns, these sequences were divided into three groups: those containing the CX4 CX2 CXn C pattern (plant-type ferredoxins), those with the CX5 CX2 CXn C pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster-binding patterns: 88 sequences had the CX4 CX2 CX29 C pattern, and 29 had the CX4 CX2 CXm C (m ≠ 29) pattern. The structures of the 88 ferredoxins with the CX4 CX2 CX29 C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 Å of the backbone root-mean-square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three-dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent-accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of proteinprotein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4 CX2 CXm C (m ≠ 29) cluster-binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup. Proteins 2002;46:110127. © 2001 Wiley-Liss, Inc.- Published
- 2002
- Full Text
- View/download PDF
111. Glucosylation of Isatin-3-Oxime followed by 2Din situ NMR in Plant Cells at Highest Magnetic Field without Labelling
- Author
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Hinse, Christiane, Unger, Matthias, Provenzani, Alessandro, and Stöckigt, Joachim
- Abstract
The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gradient assisted NMR spectroscopy in plant cell suspension cultures of Rauvolfia serpentina without labelling. The applied high magnetic field of 800 MHz allowed measurements within 20 min at concentrations of 1 of 5.76 mM. Complete glucosylation of 1 occurs inside the cells within 72 hours. During this time isatin-3-oxime-glucoside (2) accumulates without further metabolism.
- Published
- 2001
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112. Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in Charcot-Marie-Tooth type 2 neuropathies.
- Author
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Sisto, Angela, van Wermeskerken, Tamira, Pancher, Michael, Gatto, Pamela, Asselbergh, Bob, Assunção Carreira, Ágata Sofia, De Winter, Vicky, Adami, Valentina, Provenzani, Alessandro, and Timmerman, Vincent
- Subjects
- *
INDUCED pluripotent stem cells , *HEAT shock proteins , *AUTOPHAGY , *NEURAL circuitry , *MOTOR neurons - Abstract
HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1P182L and HSPB8K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
113. Additional file 2: of EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition
- Author
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Zucal, Chiara, D’Agostino, Vito, Casini, Antonio, Mantelli, Barbara, Natthakan Thongon, Soncini, Debora, Caffa, Irene, Cea, Michele, Ballestrero, Alberto, Quattrone, Alessandro, Indraccolo, Stefano, Nencioni, Alessio, and Provenzani, Alessandro
- Subjects
3. Good health - Abstract
Luciferase assays. A) Light units, normalized to protein concentration, of RLuc-cMyc 5′UTR IRES-FLuc reporter vector transduced in Jurkat cells with lentiviral particles after 48 h of treatment with or without (Mock) the indicated concentration of FK866. Two hour treatment with 250 nM of Torin 1 served as a positive control for IRES-dependent protein translation (p-value
114. [Untitled]
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Thongon, Natthakan, Castiglioni, Ilaria, Zucal, Chiara, Latorre, Elisa, D'Agostino, Vito, Bauer, Inga, Pancher, Michael, Ballestrero, Alberto, Feldmann, Georg, Nencioni, Alessio, and Provenzani, Alessandro
- Subjects
0301 basic medicine ,Regulation of gene expression ,Vimentin ,Biology ,Bioinformatics ,3. Good health ,CTGF ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,Gene expression ,Cancer research ,biology.protein ,Epithelial–mesenchymal transition ,GSK3B ,Chromatin immunoprecipitation ,Transcription factor - Abstract
// Natthakan Thongon 1, * , Ilaria Castiglioni 2, * , Chiara Zucal 1 , Elisa Latorre 1 , Vito D’Agostino 1 , Inga Bauer 3 , Michael Pancher 4 , Alberto Ballestrero 3 , Georg Feldmann 5 , Alessio Nencioni 3 , Alessandro Provenzani 1 1 Laboratory of Genomic Screening, Centre for Integrative Biology, University of Trento, Trento, Italy 2 Laboratory of Gene Expression and Muscular Dystrophy, San Raffaele Scientific Institute, Milan, Italy 3 Department of Internal Medicine, University of Genoa, Genoa, Italy 4 High Throughput Screening Facility, Centre for Integrative Biology, University of Trento, Trento, Italy 5 Laboratory of Pancreatic Cancer Translational Research, Clinic University of Bonn, Bonn, Germany * These authors have contributed equally to this work Correspondence to: Alessandro Provenzani, email: alessandro.provenzani@unitn.it Keywords: YAP, EMT, CTGF, PDAC, bisindolylmaleimides Received: August 28, 2015 Accepted: March 06, 2016 Published: March 28, 2016 ABSTRACT The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3β, caused YAP accumulation into the nucleus. Activation of β-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3β pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3β by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro , phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin , vimentin , CTGF and of the newly identified target, CD133 . In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.
115. Additional file 2: of EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition
- Author
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Zucal, Chiara, D’Agostino, Vito, Casini, Antonio, Mantelli, Barbara, Natthakan Thongon, Soncini, Debora, Caffa, Irene, Cea, Michele, Ballestrero, Alberto, Quattrone, Alessandro, Indraccolo, Stefano, Nencioni, Alessio, and Provenzani, Alessandro
- Subjects
3. Good health - Abstract
Luciferase assays. A) Light units, normalized to protein concentration, of RLuc-cMyc 5′UTR IRES-FLuc reporter vector transduced in Jurkat cells with lentiviral particles after 48 h of treatment with or without (Mock) the indicated concentration of FK866. Two hour treatment with 250 nM of Torin 1 served as a positive control for IRES-dependent protein translation (p-value
116. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage
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Becattini, Barbara, Zani, , Fabio, Breasson, Ludovic, Sardi, Claudia, D'Agostino, Vito Giuseppe, Choo, Min-Kyung, Provenzani, Alessandro, Park, Jin Mo, Solinas, Giovanni, Becattini, Barbara, Zani, , Fabio, Breasson, Ludovic, Sardi, Claudia, D'Agostino, Vito Giuseppe, Choo, Min-Kyung, Provenzani, Alessandro, Park, Jin Mo, and Solinas, Giovanni
- Abstract
Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1−/−) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1−/− mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.— Becattini, B., Zani, F., Breasson, L., Sardi, C., D’Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.
117. Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.
- Author
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D'Agostino, Vito Giuseppe, Lal, Preet, Mantelli, Barbara, Tiedje, Christopher, Zucal, Chiara, Thongon, Natthakan, Gaestel, Matthias, Latorre, Elisa, Marinelli, Luciana, Seneci, Pierfausto, Amadio, Marialaura, and Provenzani, Alessandro
- Subjects
TRANSCRIPTION factors ,GENE expression ,CARRIER proteins ,TUMOR necrosis factors ,MESSENGER RNA - Abstract
Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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118. Impact of ETV7 on chemoresistance and cancer stem-like cell plasticity in breast cancer
- Author
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Pezzè, Laura, Ciribilli, Yari, and Provenzani, Alessandro
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Area 05 - Scienze biologiche - Abstract
ETV7 is a poorly characterized transcriptional repressor that belongs to the large family of ETS transcription factors, whose members have been associated with several cancer-related processes. ETV7 is a well-recognized Interferon-stimulated gene (ISG), and it was shown that its expression can be synergistically induced by the combined treatment with the chemotherapeutic drug Doxorubicin and the inflammatory cytokine TNFa in different cancer cell lines, including the breast cancer-derived MCF7 cells. Recently, it has been shown that ETV7 expression is significantly increased in breast cancer tissues, compared to the normal breast; however, the roles and the impact of ETV7 expression in breast cancer have still to be elucidated. This project aimed at understanding the effects caused by increased ETV7 expression on breast cancer (BC) progression and resistance to conventional anti-cancer drugs. We first observed that ETV7 expression can be induced by different stimuli, particularly by the treatment with several chemotherapeutic drugs able to induce DNA damage. We also demonstrated that the expression of ETV7 could affect the sensitivity of BC cell lines to standard anti-cancer therapies, such as Doxorubicin, 5-Fluorouracil and radiotherapy, and this evidence was correlated with an increase in ABC transporters and anti-apoptotic proteins expression. By investigating the possible mechanism responsible for ETV7-dependent Doxorubicin resistance we identified a novel target gene of ETV7, DNAJC15, which is a co-chaperone protein whose repression was previously associated with drug resistance. Given the ability of cancer stem cells (CSCs) to be more chemoresistant, we analyzed the effects of ETV7 expression on the sub-population of breast CSCs. We found that ETV7 expression could exert a strong effect on breast cancer cells stemness, confirmed by both an increase in CD44+/CD24low population and mammosphere formation efficiency. In order to investigate the mechanisms responsible for these effects, we performed an RNA-seq analysis, which revealed significant repression of a signature of Interferon-stimulated genes, suggesting a possible negative feedback mechanism in the regulation of the response to Interferon. Finally, prolonged treatment of breast cancer cells with IFNb was able to rescue the effects on CSCs content. Taken collectively, our data revealed that ETV7 can affect the sensitivity of breast cancer cells to some chemotherapeutic drugs and we propose ETV7 as an important contributor to the tumor-initiating capabilities of BC cells.
- Published
- 2019
119. Characterization of Small Molecules Inhibiting the RNA Binding Protein HuR
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Lal, Preet and Provenzani, Alessandro
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BIO/11 BIOLOGIA MOLECOLARE - Abstract
HuR, the ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds to AREs (AU-rich elements) and mainly stabilizes ARE-containing mRNAs, e.g. TNFα, VEGF, c-FOS, favoring specific protein translation. TNFα mRNA is one of the most important target mRNA of HuR since the protein encoded by this gene mediates the inflammatory response and its overexpression is correlated with autoimmune diseases and cancer-related inflammation. Specific drugs are already available that can inhibit TNFα protein but cause important side-effects, as insurgence of tumoral pathologies, due to high immunodepression. Therefore, inhibition of TNFα mRNA translation by specific inhibitors targeting HuR, only in those cells undergoing pathological anomalies, is an alternative, intriguing novel therapeutic approach that deserves investigation. By REMSA and AlphaScreen assays we identified a family of low molecular weight inhibitors, called Tanshinones, among which DHTS-I (Dihydrotanshinone – I) was the most potent. Tanshinones are well known in the traditional Chinese Medicine Practice, and these anti-inflammatory agents possess the ability to prevent HuR-RNA complex formation in vitro. We further identified structural determinants of HuR and DHTS interaction using RRM1&RRM2 tandem domains. EMSA and AlphaScreen experiments, with truncated ΔRRM1 and mutants revealed that DHTS is a competitive binder of HuR with respect of target RNA. To ameliorate the solubility of DHTS, we synthesized a number of DHTS analogs, of which the most potent and soluble compound was named MFM49. We evaluated the anti-inflammatory potential of DHTS and DHTS analogs and the HuR-dependent mechanism of action, revealing that, at least in part, DHTS and DHTS analogs rely on HuR to exert their mechanism of action. Influence on NF-kB activation by DHTS and MFM49 upon LPS co-stimulation was not seen in immunofluorescence studies. So here, we disclose a previously unrecognized molecular mechanism of action exerted by DHTS, and anti-inflammatory potential of DHTS analogs opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer like anomalies.
- Published
- 2017
120. Molecular Mechanisms and Insights into the Nampt Inhibitor (FK866) Resistance in Cancers
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Thongon, Natthakan and Provenzani, Alessandro
- Subjects
BIO/11 BIOLOGIA MOLECOLARE - Abstract
Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, is one of the major factors regulating cell metabolism and NAMPT is considered a promising target for treating cancer. The NAMPT inhibitor (FK866) has exhibited anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we showed that FK866 induced translation arrest through the activation of 5’AMP-activated protein kinase (AMPK), inhibition of mTOR/4EBP1 signaling, and phosphorylation of EIF2a in leukemia cells. Cancer drug resistance continues to be a major challenge in medical oncology. Deregulated cellular metabolism is linked to such cell resistance. Indeed, both components of the glycolytic and mitochondrial pathways are involved in altered metabolism conferring chemoresistance in several cancers. In this study, we developed FK866-resistant models in T- lymphoblastic leukemia (CCRF-CEM) and breast cancer (MDA-MB231) cell lines to investigate the molecular mechanism of pharmacoresistance to NAMPT inhibitor (FK866). Our resistant cells were not inhibited at the translational level by FK866 and the drug-induced metabolic adaptations of the resistant cells conferred an advantage to counteract FK866 toxicity. We reveal a molecular mechanism by which FK866 resistant CCRF-CEM cells utilize alternative sources for NAD production to fuel cell metabolism, and metabolic reprogramming was associated to the drug resistance. Importantly, the FK866- induced metabolic alteration was overcome by the co-administration of FK866 with compounds targeting metabolism, thereby rendering a synergistic outcome and restoring cell susceptibility towards FK866. We highlighted a molecular target that favors acquiring of resistance in leukemia and breast cancer cells. In conclusion, targeting metabolic alterations associated with drug resistance to FK866 may open up unexplored opportunities for the development of new therapeutic strategies as a combinatorial treatment for cancer.
- Published
- 2017
121. Prognostic Impact of Diabetes and Prediabetes on Survival Outcomes in Patients With Chronic Heart Failure: A Post-Hoc Analysis of the GISSI-HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca-Heart Failure) Trial
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Marco Dauriz, Giovanni Targher, Pier Luigi Temporelli, Donata Lucci, Lucio Gonzini, Gian Luigi Nicolosi, Roberto Marchioli, Gianni Tognoni, Roberto Latini, Franco Cosmi, Luigi Tavazzi, Aldo Pietro Maggioni, Simona Barlera, Maria Grazia Franzosi, Aldo P. Maggioni, Maurizio Porcu, Salim Yusuf, Fulvio Camerini, Jay N. Cohn, Adriano Decarli, Bertram Pitt, Peter Sleight, Philip A. Poole‐Wilson, Enrico Geraci, Marino Scherillo, Gianna Fabbri, Barbara Bartolomei, Daniele Bertoli, Franco Cobelli, Claudio Fresco, Antonietta Ledda, Giacomo Levantesi, Cristina Opasich, Franco Rusconi, Gianfranco Sinagra, Fabio Turazza, Alberto Volpi, Martina Ceseri, Gianluca Alongi, Antonio Atzori, Filippo Bambi, Desiree Bastarolo, Francesca Bianchini, Iacopo Cangioli, Vittoriana Canu, Concetta Caporusso, Gabriele Cenni, Laura Cintelli, Michele Cocchio, Alessia Confente, Eva Fenicia, Giorgio Friso, Marco Gianfriddo, Gianluca Grilli, Beatrice Lazzaro, Giuseppe Lonardo, Alessia Luise, Rachele Nota, Mariaelena Orlando, Rosaria Petrolo, Chiara Pierattini, Valeria Pierota, Alessandro Provenzani, Velia Quartuccio, Anna Ragno, Chiara Serio, Alvise Spolaor, Arianna Tafi, Elisa Tellaroli, Stefano Ghio, Elisa Ghizzardi, Serge Masson, Lella Crociati, Maria Teresa La Rovere, Ugo Corrà, Andrea Finzi, Marco Gorini, Valentina Milani, Giampietro Orsini, Elisa Bianchini, Silvia Cabiddu, Ilaria Cangioli, Laura Cipressa, Maria Lucia Cipressa, Giuseppina Di Bitetto, Barbara Ferri, Luisa Galbiati, Andrea Lorimer, Carla Pera, Paola Priami, Antonella Vasamì, T. Moccetti, M.G. Rossi, E. Pasotti, F. Vaghi, P. Roncarolo, M.T. Zunino, F. Matta, E. Actis Perinetto, F. Gaita, G. Azzaro, M. Zanetta, A.M. Paino, U. Parravicini, D. Vegis, R. Conte, P. Ferraro, A. De Bernardi, S. Morelloni, M. Fagnani, P. Greco Lucchina, L. Montagna, E. Bellone, D. Sappè, F. Ferraro, M. Delucchi, S.G. Reynaud, M. Dore, A. La Brocca, N. Massobrio, L. Bo, R. Trinchero, M. Imazio, G. Brocchi, A. Nejrotti, L. Rissone, S. Gabasio, C. Zocchi, S. Randazzo, A. Crenna, P. Giannuzzi, E. Bonanomi, A. Mezzani, M. De Marchi, G. Begliuomini, C.A. Gianonatti, A. Gavazzi, A. Grosu, L. Dei Cas, S. Nodari, P. Garyfallidis, A. Bertoletti, C. Bonifazi, S. Arisi, F. Mascaro, M. Fraccarollo, S. Dell'Orto, M. Sfolcini, F. Bortolini, D. Raccagni, A. Turelli, M. Santarone, E. Miglierina, L. Sormani, R. Jemoli, F. Tettamanti, S. Pirelli, C. Bianchi, S. Verde, M. Mariani, V. Ziacchi, A. Ferrazza, A. Russo, M. Bortolotti, G.F. Pasini, A. Volpi, K.N. Jones, D. Cuzzucrea, G. Gullace, C. Carbone, A. Granata, S. De Servi, G. Del Rosso, C. Inserra, E. Renaldini, C. Zappa, M. Moretti, R. Zanini, M. Ferrari, E. Moroni, A. Cei, C. Lissi, E. Dovico, C. Fiorentini, P. Palermo, B. Brusoni, M. Negrini, J. Heyman, G.B. Danzi, A. Finzi, M. Frigerio, F. Turazza, L. Beretta, A. Sachero, F. Casazza, L. Squadroni, F. Lombardi, L. Marano, A. Margonato, G. Fragasso, O.C. Febo, E. Aiolfi, F. Olmetti, A. Grieco, V. Antonazzo, G. Specchia, A. Mortara, F. Robustelli, M.G. Songini, C. Schweiger, A. Frisinghelli, M. Palvarini, C. Campana, L. Scelsi, N. Ajmone Marsan, F. Cobelli, A. Gualco, C. Opasich, S. De Feo, R. Mazzucco, M.A. Iannone, T. Diaco, D. Zaniboni, G. Milanesi, D. Nassiacos, S. Meloni, P. Giani, T. Nicoli, C. Malinverni, A. Gusmini, L. Pozzoni, G. Bisiani, P. Margaroli, A. Schizzarotto, A. Daverio, G. Occhi, N. Partesana, P. Bandini, M.G. Rosella, S. Giustiniani, G. Cucchi, R. Pedretti, R. Raimondo, R. Vaninetti, A. Fedele, I. Ghezzi, E. Rezzonico, J.A. Salerno Uriarte, F. Morandi, F. Salvucci, C. Valenti, G. Graziano, M. Romanò, C. Cimminiello, I. Mangone, M. Lombardo, P. Quorso, G. Marinoni, M. Breghi, M. Erckert, A. Dienstl, G. Mirante Marini, C. Stefenelli, G. Cioffi, E. Buczkowska, A. Bonanome, F. Bazzanini, L. Parissenti, C. Serafini, G. Catania, L. Tarantini, G. Rigatelli, S. Boni, A. Pasini, E. Masini, A.A. Zampiero, M. Zanchetta, L. Franceschetto, P. Delise, C. Marcon, A. Sacchetta, L. Borgese, L. Artusi, P. Casolino, F. Corbara, A. Banzato, M. Barbiero, M.P. Aldegheri, R. Bazzucco, G. Crivellenti, A. Raviele, C. Zanella, P. Pascotto, P. Sarto, S. Milan, E. Barbieri, P. Girardi, W. Dalla Villa, J. Dalle Mule, M.L. Di Sipio, R. Cazzin, D. Milan, P. Zonzin, M. Carraro, R. Rossi, E. Carbonieri, I. Rossi, P. Stritoni, P. Meneghetti, G. Risica, P.L. Tenderini, C. Vassanelli, L. Zanolla, G. Perini, G. Brighetti, R. Chiozza, G. Giuliano, R. Gortan, R. Cesanelli, G.L. Nicolosi, R. Piazza, L. Mos, O. Vriz, D. Pavan, G. Pascottini, E. Alberti, M. Werren, L. Solinas, G. Sinagra, F. Longaro, P. Fioretti, M.C. Albanese, D. Miani, R. Gianrossi, A. Pende, P. Rubartelli, O. Magaia, S. Domenicucci, D. Caruso, A.S. Faraguti, L. Magliani, F. Miccoli, G. Guglielmino, D. Bertoli, A. Cantarelli, S. Orlandi, A. Vallebona, A. Pozzati, G. Brega, L.G. Pancaldi, R. Vandelli, S. Urbinati, M.G. Poci, M. Zoli, G.M. Costa, U. Guiducci, G. Zobbi, F. Tartagni, A. Tisselli, A. Gentili, P. Pieri, E. Cagnetta, S. Bendinelli, A. Barbieri, R. Conti, R. Ferrari, F. Merlini, A. Fucili, P. Moruzzi, E. Buia, M. Galvani, D. Ferrini, G. Baggioni, P. Yiannacopulu, G. Canè, A. Bonfiglioli, R. Zandomeneghi, L. Brugioni, A. Giannini, R. Di Ruvo, M. Giuliani, L. Rusconi, P. Del Corso, G. Piovaccari, F. Bologna, P. Venturi, F. Melandri, E. Bagni, L. Bolognese, R. Perticucci, A. Zuppiroli, M. Nannini, N. Consoli, P. Petrone, C. Pipitò, L. Colombi, D. Bernardi, P.R. Mariani, R. Testa, F. Mazzinghi, F. Cosmi, D. Cosmi, A. Zipoli, A. Cecchi, G. Castelli, M. Ciaccheri, F. Mori, F. Pieri, P. Valoti, D. Chiarantini, G.M. Santoro, C. Minneci, F. Marchi, M. Milli, G. Zambaldi, A.A. Brandinelli Geri, M. Cipriani, M. Alessandri, S. Severi, S. Stefanelli, A. Comella, R. Poddighe, A. Digiorgio, M. Carluccio, S. Berti, A. Rizza, V. Bonatti, V. Molendi, A. Brancato, N. D'Aprile, G. Giappichini, S. Del Vecchio, G. Mantini, F. De Tommasi, G. Meucci, M. Cordoni, S. Bechi, L. Barsotti, P. Baldini, M. Romei, G. Scopelliti, G. Lauri, F. Pestelli, F. Furiozzi, M. Cocchieri, D. Severini, F. Patriarchi, P. Chiocchi, M. Buccolieri, S. Martinelli, A. Wee, F. Angelici, M. Bernardinangeli, G. Proietti, B. Biscottini, R. Panciarola, L. Marinacci, G.P. Perna, D. Gabrielli, A. Moraca, L. Moretti, L. Partemi, G. Gregori, R. Amici, G. Patteri, P. Capone, E. Savini, G.L. Morgagni, L. Paccaloni, F. Pezzuoli, S. Carincola, S. Papi, S. De Crescentini, P. Gerardi, P. Midi, E. Gallenzi, G. Pajes, C. Mancone, V. Di Spirito, M. Di Gennaro, S. Calcagno, S. Toscano, S. Antonicoli, F. Carta, G. Giorgi, F. Comito, E. Daniele, O. Ciarla, P.G. Gelfo, A. Acquaviva, D. Testa, G. Testa, F.A. Pagliaro, F. Russo, F. Vetta, I. Marchese, G. Di Sciascio, A. D'Ambrosio, F. Leggio, D. Del Sindaco, A. Lacchè, A. Avallone, M.P. Risa, P. Azzolini, E. Baldo, E. Giovannini, G. Pulignano, C. Tondo, E. Picchio, E. ani, P. Tanzi, F. Pozzar, F. Farnetti, M. Azzarito, M. Santini, A. Varveri, G. Ferraiuolo, C. Valtorta, A. Gaspardone, G. Barbato, V. Ceci, N. Aspromonte, F. Bellocci, C. Colizzi, F. Fedele, F.I. Perez, A. Galati, A. Rossetti, A. Mainella, D. etta, C. Matteucci, G. Busi, A. De Angelis, G. Farina, A. Granatelli, F. Leone, F. Frasca, R. Di Giovambattista, G. Castellani, G. Massaro, G. Mastrogiuseppe, A. Vacri, F. De Sanctis, M. Cioli, S. Di Luzio, C. Napoletano, L.L. Piccioni, G. De Simone, A. Ottaviano, V. Mazza, C. Spedaliere, D. Staniscia, E. Calgione, G. De Marco, T. Chiacchio, T. Di Napoli, S. Romanzi, G. Salvatore, P. Golino, A. Palermo, F. Mascia, A. Vetrano, A. Vinciguerra, L. Caliendo, R. Longobardi, G. De Caro, R. Di Nola, F. Piemonte, D. Prinzi, P. De Rosa, V. De Rosa, F. Riello, V. Capuano, G. Vecchio, M. Landi, S. Amato, M. Garofalo, M. D'Avino, P. Sensale, O. Maiolica, R. Santoro, P. Caso, D. Miceli, N. Maurea, U. Bianchi, C. Crispo, M. Chiariello, P. Perrone Filardi, L. Russo, N. Capuano, G. Ungaro, G. Vergara, F. Scafuro, G. D'Angelo, C. Campaniello, P. Bottiglieri, A. Volpe, R. Battista, L. De Risi, G. Cardillo, G. Sibilio, A.P. Marino, F. Silvestri, P. Predotti, A. Iervoglini, C. De Matteis, P. Sarnicola, M.M. Matarazzo, S. Baldi, V. Iuliano, C. Astarita, P. Cuccaro, A. Liguori, G. Liguori, G. Gregorio, L. Petraglia, G. Antonelli, G. Amodio, I. De Luca, D. Traversa, G. Franchini, M.L. Lenti, D. Cavallari, C. D'Agostino, G. Scalera, C.M. Altamura, M. Russo, A.R. Mascolo, G. Pettinati, S.A. Ciricugno, D. Scrutinio, A. Passantino, D. Mastrangelo, A. Di Masi, R. De Carne, M. Cannone, F. Dibiase, M. Pensato, F. Loliva, F. Trapani, I. Panettieri, L. Leone, M. Di Biase, M. Carrone, V. Gallone, F. Cocco, M. Costantini, C. Tritto, F. Cavalieri, L. Stella, F. Magliari, M. Callerame, A. De Giorgi, L. Pellegrino, M. Correra, V. Portulano, G.L. Nisi, G. Grassi, E. Cristallo, D. De Laura, C. Salerno, R. Fanelli, M. Villella, S. Pede, A. Renna, E. De Lorenzi, L. Urso, V. Lenti, A. Peluso, N. Baldi, G. Polimeni, P. Palma, R. Lauletta, E. Tagliamonte, T. Cirillo, B. Silvestri, G. Centonze, B. D'Alessandro, L. Truncellito, D. Mecca, M.A. Petruzzi, R.O.M. Coviello, A. Lopizzo, M. telli, S. Barbuzzi, S. Gubelli, G. Germinario, N. Cosentino, A. Mingrone, R. Vico, G. Borrello, M.L. Mazza, R. Cimino, D. Galasso, F. Cassadonte, U. Talarico, F. Perticone, S. Cassano, F. Catapano, S. Calemme, E. Feraco, C. Cloro, G. Misuraca, R. Caporale, L. Vigna, V. Spagnuolo, F. De Rosa, G. Spadafora, G. Zampaglione, R. Russo, F.A. Schipani, A.F. Ferragina, D. Stranieri, G. Musca, C. Carpino, P. Bencardino, F. Raimondo, D. Musacchio, G. Pulitanò, A. Ruggeri, A. Provenzano, S. Salituri, M. Musolino, S. Calandruccio, A. Marrari, E. Tripodi, R. Scali, L. Anastasio, A. Arone, P. Aragona, L. Donnangelo, M.G.A. Comito, F. Bilotta, I. Vaccaro, R. Rametta, V. Ventura, A. Bonvegna, A. Alì, C. Cinnirella, M. Raineri, F. Pompeo, N. Cascio Ingurgio, V. Carini, R. Coco, G. Giunta, G. Leonardi, V. Randazzo, V. Di Blasi, C. Tamburino, G. Russo, S. Mangiameli, R. Cardillo, D. Castelli, V. Inserra, A. Arena, M.M. Gulizia, S. Raciti, G. Rapisarda, R. Romano, P. Prestifilippo, G.B. Braschi, G. Ledda, R. Terrazzino, M. De Caro, G. Scilabra, B. agnino, R. Grassi, G. Di Tano, G.F. Scimone, L. Vasquez, C. Coppolino, A. Casale, M. Castelli, G. D'Urso, E. D'Antonio, L. Lo Presti, E. Badalamenti, P. Conti, N. Sanfilippo, V. Cirrincione, M.T. Cinà, G. Cusimano, A. Taormina, P. Giuliano, A. Bajardi, V. Mandalà, A. Canonico, G. Geraci, F.P. Sabella, F. Enia, A.M. Floresta, I. Lo Cascio, D. Gumina, A. Cavallaro, G. Piccione, R. Ferrante, M. Blandino, M.S. Iudicello, E. Mossuti, G. Romano, L. Lombardo, P. Monastra, D. Di Vincenzo, M. Porcu, P. Orrù, F. Muscas, G. Giardina, M. Corda, G. Locci, A. Podda, M. Ledda, P. Siddi, C. Lai, G. Pili, G. Mercuro, G. Mureddu, A. Ganau, G. Meloni, G. Poddighe, G. Sanna, Dauriz, Marco, Targher, Giovanni, Temporelli, Pier Luigi, Lucci, Donata, Gonzini, Lucio, Nicolosi, Gian Luigi, Marchioli, Roberto, Tognoni, Gianni, Latini, Roberto, Cosmi, Franco, Tavazzi, Luigi, Maggioni, Aldo Pietro, on behalf of the GISSI-HF, Investigator, Margonato, Alberto, Moccetti, T., Rossi, M. G., Pasotti, E., Vaghi, F., Roncarolo, P., Zunino, M. T., Matta, F., Actis Perinetto, E., Gaita, F., Azzaro, G., Zanetta, M., Paino, A. M., Parravicini, U., Vegis, D., Conte, R., Ferraro, P., De Bernardi, A., Morelloni, S., Fagnani, M., Greco Lucchina, P., Montagna, L., Bellone, E., Sappè, D., Ferraro, F., Delucchi, M., Reynaud, S. G., Dore, M., La Brocca, A., Massobrio, N., Bo, L., Trinchero, R., Imazio, M., Brocchi, G., Nejrotti, A., Rissone, L., Gabasio, S., Zocchi, C., Randazzo, S., Crenna, A., Giannuzzi, P., Bonanomi, E., Mezzani, A., De Marchi, M., Begliuomini, G., Gianonatti, C. A., Gavazzi, A., Grosu, A., Dei Cas, L., Nodari, S., Garyfallidis, P., Bertoletti, A., Bonifazi, C., Arisi, S., Mascaro, F., Fraccarollo, M., Dell'Orto, S., Sfolcini, M., Bortolini, F., Raccagni, D., Turelli, A., Santarone, M., Miglierina, E., Sormani, L., Jemoli, R., Tettamanti, F., Pirelli, S., Bianchi, C., Verde, S., Mariani, M., Ziacchi, V., Ferrazza, A., Russo, A., Bortolotti, M., Pasini, G. F., Volpi, A., Jones, K. N., Cuzzucrea, D., Gullace, G., Carbone, C., Granata, A., De Servi, S., Del Rosso, G., Inserra, C., Renaldini, E., Zappa, C., Moretti, M., Zanini, R., Ferrari, M., Moroni, E., Cei, A., Lissi, C., Dovico, E., Fiorentini, C., Palermo, P., Brusoni, B., Negrini, M., Heyman, J., Danzi, G. 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A., Cipriani, M., Alessandri, M., Severi, S., Stefanelli, S., Comella, A., Poddighe, R., Digiorgio, A., Carluccio, M., Berti, S., Rizza, A., Bonatti, V., Molendi, V., Brancato, A., D'Aprile, N., Giappichini, G., Del Vecchio, S., Mantini, G., De Tommasi, F., Meucci, G., Cordoni, M., Bechi, S., Barsotti, L., Baldini, P., Romei, M., Scopelliti, G., Lauri, G., Pestelli, F., Furiozzi, F., Cocchieri, M., Severini, D., Patriarchi, F., Chiocchi, P., Buccolieri, M., Martinelli, S., Wee, A., Angelici, F., Bernardinangeli, M., Proietti, G., Biscottini, B., Panciarola, R., Marinacci, L., Perna, G. P., Gabrielli, D., Moraca, A., Moretti, L., Partemi, L., Gregori, G., Amici, R., Patteri, G., Capone, P., Savini, E., Morgagni, G. L., Paccaloni, L., Pezzuoli, F., Carincola, S., Papi, S., De Crescentini, S., Gerardi, P., Midi, P., Gallenzi, E., Pajes, G., Mancone, C., Di Spirito, V., Di Gennaro, M., Calcagno, S., Toscano, S., Antonicoli, S., Carta, F., Giorgi, G., Comito, F., Daniele, E., Ciarla, O., Gelfo, P. G., Acquaviva, A., Testa, D., Testa, G., Pagliaro, F. A., Russo, F., Vetta, F., Marchese, I., Di Sciascio, G., D'Ambrosio, A., Leggio, F., Del Sindaco, D., Lacchè, A., Avallone, A., Risa, M. P., Azzolini, P., Baldo, E., Giovannini, E., Pulignano, G., Tondo, C., Picchio, E., Biffani, E., Tanzi, P., Pozzar, F., Farnetti, F., Azzarito, M., Santini, M., Varveri, A., Ferraiuolo, G., Valtorta, C., Gaspardone, A., Barbato, G., Ceci, V., Aspromonte, N., Bellocci, F., Colizzi, C., Fedele, F., Perez, F. I., Galati, A., Rossetti, A., Mainella, A., Ciuffetta, D., Matteucci, C., Busi, G., De Angelis, A., Farina, G., Granatelli, A., Leone, F., Frasca, F., Di Giovambattista, R., Castellani, G., Massaro, G., Mastrogiuseppe, G., Vacri, A., De Sanctis, F., Cioli, M., Di Luzio, S., Napoletano, C., Piccioni, L. L., De Simone, G., Ottaviano, A., Mazza, V., Spedaliere, C., Staniscia, D., Calgione, E., De Marco, G., Chiacchio, T., Di Napoli, T., Romanzi, S., Salvatore, G., Golino, P., Palermo, A., Mascia, F., Vetrano, A., Vinciguerra, A., Caliendo, L., Longobardi, R., De Caro, G., Di Nola, R., Piemonte, F., Prinzi, D., De Rosa, P., De Rosa, V., Riello, F., Capuano, V., Vecchio, G., Landi, M., Amato, S., Garofalo, M., D'Avino, M., Sensale, P., Maiolica, O., Santoro, R., Caso, P., Miceli, D., Maurea, N., Bianchi, U., Crispo, C., Chiariello, M., Perrone Filardi, P., Russo, L., Capuano, N., Ungaro, G., Vergara, G., Scafuro, F., D'Angelo, G., Campaniello, C., Bottiglieri, P., Volpe, A., Battista, R., De Risi, L., Cardillo, G., Sibilio, G., Marino, A. P., Silvestri, F., Predotti, P., Iervoglini, A., De Matteis, C., Sarnicola, P., Matarazzo, M. M., Baldi, S., Iuliano, V., Astarita, C., Cuccaro, P., Liguori, A., Liguori, G., Gregorio, G., Petraglia, L., Antonelli, G., Amodio, G., De Luca, I., Traversa, D., Franchini, G., Lenti, M. L., Cavallari, D., D'Agostino, C., Scalera, G., Altamura, C. M., Russo, M., Mascolo, A. R., Pettinati, G., Ciricugno, S. A., Scrutinio, D., Passantino, A., Mastrangelo, D., Di Masi, A., De Carne, R., Cannone, M., Dibiase, F., Pensato, M., Loliva, F., Trapani, F., Panettieri, I., Leone, L., Di Biase, M., Carrone, M., Gallone, V., Cocco, F., Costantini, M., Tritto, C., Cavalieri, F., Stella, L., Magliari, F., Callerame, M., De Giorgi, A., Pellegrino, L., Correra, M., Portulano, V., Nisi, G. L., Grassi, G., Cristallo, E., De Laura, D., Salerno, C., Fanelli, R., Villella, M., Pede, S., Renna, A., De Lorenzi, E., Urso, L., Lenti, V., Peluso, A., Baldi, N., Polimeni, G., Palma, P., Lauletta, R., Tagliamonte, E., Cirillo, T., Silvestri, B., Centonze, G., D'Alessandro, B., Truncellito, L., Mecca, D., Petruzzi, M. A., Coviello, R. O. M., Lopizzo, A., Chiaffitelli, M., Barbuzzi, S., Gubelli, S., Germinario, G., Cosentino, N., Mingrone, A., Vico, R., Borrello, G., Mazza, M. L., Cimino, R., Galasso, D., Cassadonte, F., Talarico, U., Perticone, F., Cassano, S., Catapano, F., Calemme, S., Feraco, E., Cloro, C., Misuraca, G., Caporale, R., Vigna, L., Spagnuolo, V., De Rosa, F., Spadafora, G., Zampaglione, G., Russo, R., Schipani, F. A., Ferragina, A. F., Stranieri, D., Musca, G., Carpino, C., Bencardino, P., Raimondo, F., Musacchio, D., Pulitanò, G., Ruggeri, A., Provenzano, A., Salituri, S., Musolino, M., Calandruccio, S., Marrari, A., Tripodi, E., Scali, R., Anastasio, L., Arone, A., Aragona, P., Donnangelo, L., Comito, M. G. A., Bilotta, F., Vaccaro, I., Rametta, R., Ventura, V., Bonvegna, A., Alì, A., Cinnirella, C., Raineri, M., Pompeo, F., Cascio Ingurgio, N., Carini, V., Coco, R., Giunta, G., Leonardi, G., Randazzo, V., Di Blasi, V., Tamburino, C., Russo, G., Mangiameli, S., Cardillo, R., Castelli, D., Inserra, V., Arena, A., Gulizia, M. M., Raciti, S., Rapisarda, G., Romano, R., Prestifilippo, P., Braschi, G. B., Ledda, G., Terrazzino, R., De Caro, M., Scilabra, G., Graffagnino, B., Grassi, R., Di Tano, G., Scimone, G. F., Vasquez, L., Coppolino, C., Casale, A., Castelli, M., D'Urso, G., D'Antonio, E., Lo Presti, L., Badalamenti, E., Conti, P., Sanfilippo, N., Cirrincione, V., Cinà, M. T., Cusimano, G., Taormina, A., Giuliano, P., Bajardi, A., Mandalà, V., Canonico, A., Geraci, G., Sabella, F. P., Enia, F., Floresta, A. M., Lo Cascio, I., Gumina, D., Cavallaro, A., Piccione, G., Ferrante, R., Blandino, M., Iudicello, M. S., Mossuti, E., Romano, G., Lombardo, L., Monastra, P., Di Vincenzo, D., Porcu, M., Orrù, P., Muscas, F., Giardina, G., Corda, M., Locci, G., Podda, A., Ledda, M., Siddi, P., Lai, C., Pili, G., Mercuro, G., Mureddu, G., Ganau, A., Meloni, G., Poddighe, G., Sanna, G., Barlera, Simona, Franzosi, Maria Grazia, Porcu, Maurizio, Yusuf, Salim, Camerini, Fulvio, Cohn, Jay N., Decarli, Adriano, Pitt, Bertram, Sleight, Peter, Poole-Wilson, Philip A., Geraci, Enrico, Scherillo, Marino, Fabbri, Gianna, Bartolomei, Barbara, Bertoli, Daniele, Cobelli, Franco, Fresco, Claudio, Ledda, Antonietta, Levantesi, Giacomo, Opasich, Cristina, Rusconi, Franco, Sinagra, Gianfranco, Turazza, Fabio, Volpi, Alberto, Ceseri, Martina, Alongi, Gianluca, Atzori, Antonio, Bambi, Filippo, Bastarolo, Desiree, Bianchini, Francesca, Cangioli, Iacopo, Canu, Vittoriana, Caporusso, Concetta, Cenni, Gabriele, Cintelli, Laura, Cocchio, Michele, Confente, Alessia, Fenicia, Eva, Friso, Giorgio, Gianfriddo, Marco, Grilli, Gianluca, Lazzaro, Beatrice, Lonardo, Giuseppe, Luise, Alessia, Nota, Rachele, Orlando, Mariaelena, Petrolo, Rosaria, Pierattini, Chiara, Pierota, Valeria, Provenzani, Alessandro, Quartuccio, Velia, Ragno, Anna, Serio, Chiara, Spolaor, Alvise, Tafi, Arianna, Tellaroli, Elisa, Ghio, Stefano, Ghizzardi, Elisa, Masson, Serge, Crociati, Lella, La Rovere, Maria Teresa, Corrà, Ugo, Di Giulio, Paola, Finzi, Andrea, Gorini, Marco, Milani, Valentina, Orsini, Giampietro, Bianchini, Elisa, Cabiddu, Silvia, Cangioli, Ilaria, Cipressa, Laura, Cipressa, Maria Lucia, Di Bitetto, Giuseppina, Ferri, Barbara, Galbiati, Luisa, Lorimer, Andrea, Pera, Carla, Priami, Paola, and Vasamì, Antonella
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Blood Glucose ,Male ,Glycated Hemoglobin A ,heart failure ,Kaplan-Meier Estimate ,prediabetes ,030204 cardiovascular system & hematology ,time factors ,Settore MED/11 ,cause of death ,0302 clinical medicine ,Glycemic control ,prediabetic state ,Cause of Death ,italy ,middle aged ,Prevalence ,80 and over ,double-blind method ,blood glucose ,risk factors ,030212 general & internal medicine ,Prediabetes ,Rosuvastatin Calcium ,humans ,rosuvastatin calcium ,Cause of death ,Original Research ,Metabolic Syndrome ,Aged, 80 and over ,adult ,Chronic heart failure ,Diabetes mellitus ,Heart failure ,Mortality ,Cardiology and Cardiovascular Medicine ,Hazard ratio ,chronic heart failure ,diabetes mellitus ,glycemic control ,mortality ,Treatment Outcome ,Adolescent ,Biomarkers ,Chronic Disease ,Diabetes Mellitus ,Fatty Acids, Omega-3 ,Double-Blind Method ,Hydroxymethylglutaryl-CoA Reductase Inhibitors ,Hospitalization ,Heart Failure ,Italy ,Prediabetic State ,Risk Assessment ,Proportional Hazards Models ,Risk Factors ,Time Factors ,risk assessment ,Middle Aged ,kaplan-meier estimate ,aged ,female ,Prediabete ,young adult ,Female ,omega-3 ,Human ,hospitalization ,Adult ,medicine.medical_specialty ,Diabetes mellitu ,proportional hazards models ,Time Factor ,hydroxymethylglutaryl-coa reductase inhibitors ,prevalence ,fatty acids ,03 medical and health sciences ,Young Adult ,male ,Internal medicine ,Post-hoc analysis ,glycated hemoglobin a ,medicine ,Intensive care medicine ,Aged ,Glycated Hemoglobin ,Proportional hazards model ,business.industry ,Risk Factor ,biomarkers ,Biomarker ,medicine.disease ,Clinical trial ,adolescent ,Proportional Hazards Model ,treatment outcome ,aged, 80 and over ,chronic disease ,fatty acids, omega-3 ,cardiology and cardiovascular medicine ,Hydroxymethylglutaryl-CoA Reductase Inhibitor ,business - Abstract
Background The independent prognostic impact of diabetes mellitus ( DM ) and prediabetes mellitus (pre‐ DM ) on survival outcomes in patients with chronic heart failure has been investigated in observational registries and randomized, clinical trials, but the results have been often inconclusive or conflicting. We examined the independent prognostic impact of DM and pre‐ DM on survival outcomes in the GISSI ‐HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca‐Heart Failure) trial. Methods and Results We assessed the risk of all‐cause death and the composite of all‐cause death or cardiovascular hospitalization over a median follow‐up period of 3.9 years among the 6935 chronic heart failure participants of the GISSI ‐ HF trial, who were stratified by presence of DM (n=2852), pre‐ DM (n=2013), and non‐ DM (n=2070) at baseline. Compared with non‐ DM patients, those with DM had remarkably higher incidence rates of all‐cause death (34.5% versus 24.6%) and the composite end point (63.6% versus 54.7%). Conversely, both event rates were similar between non‐ DM patients and those with pre‐ DM . Cox regression analysis showed that DM , but not pre‐ DM , was associated with an increased risk of all‐cause death (adjusted hazard ratio, 1.43; 95% CI , 1.28–1.60) and of the composite end point (adjusted hazard ratio, 1.23; 95% CI , 1.13–1.32), independently of established risk factors. In the DM subgroup, higher hemoglobin A1c was also independently associated with increased risk of both study outcomes (all‐cause death: adjusted hazard ratio, 1.21; 95% CI , 1.02–1.43; and composite end point: adjusted hazard ratio, 1.14; 95% CI , 1.01–1.29, respectively). Conclusions Presence of DM was independently associated with poor long‐term survival outcomes in patients with chronic heart failure. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 00336336.
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- 2017
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122. Regulation of HuR structure and function by dihydrotanshinone-I
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Ranjan Preet, Luciana Marinelli, Pierfausto Seneci, Emiliano Biasini, Linda Cerofolini, Ettore Novellino, Vito Giuseppe D'Agostino, Dan A. Dixon, Vikalp Vishwakarma, Isabelle Bonomo, Carmelo Fuccio, Max S. Fairlamb, Sha Neisha Williams, Marco Fragai, Preet Lal, Saioa R. Elezgarai, Rachel Munk, Alessandro Provenzani, Kotb Abdelmohsen, Chiara Zucal, Myriam Gorospe, Erik Dassi, Danilo Di Maio, Claudio Luchinat, Elin Lehrmann, Stefano Giuntini, Leonardo Manzoni, Alessandro Quattrone, Lal, Preet, Cerofolini, Linda, D'Agostino, Vito Giuseppe, Zucal, Chiara, Fuccio, Carmelo, Bonomo, Isabelle, Dassi, Erik, Giuntini, Stefano, Maio, Danilo Di, Vishwakarma, Vikalp, Preet, Ranjan, Williams, Sha Neisha, Fairlamb, Max S., Munk, Rachel, Lehrmann, Elin, Abdelmohsen, Kotb, Elezgarai, Saioa R., Luchinat, Claudio, Novellino, Ettore, Quattrone, Alessandro, Biasini, Emiliano, Manzoni, Leonardo, Gorospe, Myriam, Dixon, Dan A., Seneci, Pierfausto, Marinelli, Luciana, Fragai, Marco, and Provenzani, Alessandro
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,Xenograft Model Antitumor Assay ,Immunoprecipitation ,Protein Conformation ,Protein Domain ,3' Untranslated Region ,Protein domain ,ELAV-Like Protein 1 ,Mice, Neurologic Mutant ,RNA-binding protein ,Biology ,Molecular Dynamics Simulation ,03 medical and health sciences ,Mice, Neurologic Mutants ,Protein Domains ,Phenanthrene ,dihydrotanshinone I ,Genetics ,Animals ,Humans ,Point Mutation ,RNA, Messenger ,Furans ,3' Untranslated Regions ,Molecular Biology ,Ribonucleoprotein ,AU-rich element ,AU Rich Elements ,Three prime untranslated region ,Animal ,Quinones ,RNA ,Phenanthrenes ,Xenograft Model Antitumor Assays ,Antineoplastic Agents, Phytogenic ,Cell biology ,AU Rich Element ,030104 developmental biology ,HuR ,Human - Abstract
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
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- 2017
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123. Molecular Effects of the Nampt Inhibitor FK866 on Leukemia Cells
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Zucal, Chiara, Provenzani, Alessandro, and D'Agostino, Vito Giuseppe
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BIO/11 BIOLOGIA MOLECOLARE - Abstract
Aberrant activation of metabolic pathways has emerged as an hallmark of proliferating cancer cells and pharmaceutical approaches targeting cell metabolism hold great potential for cancer treatment. A critical factor in cellular metabolism is nicotinamide adenine dinucleotide (NAD+) and cancer cells highly rely on it to face increased metabolic demands and proliferation rates. Intracellular NAD+ is a key metabolite involved in several cellular processes, acting either as a coenzyme in redox reactions or as a substrate for NAD+-degrading enzymes such as poly (ADP-ribose) polymerases (PARPs), CD38, and sirtuins, regulating processes that undergo fundamental changes during malignant transformation. Although NAD+ can be generated de novo from tryptophan precursor, the major route of biosynthesis is through a nicotinamidesalvage process. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide in mammalian cells. A number of cancers present an increased expression of NAMPT, and high NAMPT levels have been shown to be essential to support cancer cell growth, survival and EMT transition and to correlate with adverse prognosis. NAMPT is therefore a key factor regulating tumor cell metabolism and is thus considered a promising anti-cancer target. FK866 is a specific NAMPT inhibitor that lowers NAD+ concentration in cancer cells, reducing the activity of NAD+-dependent enzymes, impacting on ATP production and promoting cell death. NAMPT inhibition was proven to be highly effective in both lymphoid and myeloid-derived hematological malignancies in preclinical studies without affecting healthy cells, such as hematopoietic stem cells. FK866 has completed a phase I trial in oncology with advanced solid tumors. Thrombocytopenia was the dose-limiting toxicity, suggesting that this drug is a good candidate for clinical applications. We investigated the mechanism of action of FK866 in T-ALL derived cell lines as well as in primary leukemia cells. FK866-induced metabolic stress and NAMPT ablation elicited a strong arrest of protein synthesis as early cell response. FK866 induced activation of the AMP-activated protein kinase (AMPK), which subsequently drove the inhibition of the mTOR/4EBP1 signaling cascade and of the major initiation factor EIF2A, impairing protein synthesis. Furthermore, FK866-induced stress reduced the levels of the anti-apoptotic protein MCL1 and impacted on the endoplasmic reticulum homeostasis. In addition, we established and characterized an FK866-resistant model derived from the T-ALL cell line Jurkat. Target-specific acquired resistance has been described after several therapies and can be modeled in vitro by growing cells in presence of increasing concentrations of drug. In our resistant cells, FK866 treatment only partially impacted on NAD+ content, whereas ATP levels were recovered and protein translation resumed. Notably, during in vitro acquisition of drug resistance, mutations in the NAMPT gene have not occurred. In the last years, many NAMPT inhibitors have been synthesized and characterized. The obtained results provide new insight into the role of the NAMPT-mediated NAD+ salvage pathway in cancer cell metabolism and the molecular mechanisms of FK866, which will be useful to formulate specific and effective combinatorial drug therapies.
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- 2016
124. Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function
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Barbara Mantelli, Natthakan Thongon, Preet Lal, Vito Giuseppe D'Agostino, Christopher Tiedje, Marialaura Amadio, Elisa Latorre, Chiara Zucal, Alessandro Provenzani, Pierfausto Seneci, Matthias Gaestel, Luciana Marinelli, D'Agostino, Vito Giuseppe, Lal, Preet, Mantelli, Barbara, Tiedje, Christopher, Zucal, Chiara, Thongon, Natthakan, Gaestel, Matthia, Latorre, Elisa, Marinelli, Luciana, Seneci, Pierfausto, Amadio, Marialaura, and Provenzani, Alessandro
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RNA Processing ,Cytoplasm ,Translational efficiency ,Messenger ,Drug Resistance ,Post-Transcriptional ,RNA-binding protein ,Breast Neoplasms ,Biology ,Bioinformatics ,Article ,Cell Line ,ELAV-Like Protein 1 ,Cell Line, Tumor ,Gene expression ,Humans ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Furans ,Regulation of gene expression ,Messenger RNA ,Tumor ,Multidisciplinary ,Tumor Necrosis Factor-alpha ,Quinones ,Drug Resistance, Neoplasm ,Female ,Gene Expression Regulation ,MCF-7 Cells ,Phenanthrenes ,Polyribosomes ,Protein Binding ,RNA-Binding Proteins ,RNA ,Translation (biology) ,Cell biology ,Cancer cell ,Neoplasm - Abstract
Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.
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- 2015
125. Development of drug screening assays for identification of new molecules against pancreatic ductal adenocarcinoma
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Castiglioni, Ilaria and Provenzani, Alessandro
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Area 05 - Scienze biologiche - Abstract
The yes-associated protein, YAP, is a transcriptional co-activator, able to regulate the expression level of a wide range of target genes. Despite deregulation of YAP has been associated with cancer etiology, no compounds are known to specifically modulate its functions. Here we identified an inhibitor of YAP functionality, called GF 10923X, and we proposed it as a potential lead molecule to be used against disease where YAP is found deregulated, pancreatic ductal adenocarcinoma (PDAC) included. GF 109203X is an already known kinase inhibitor, preferentially targeting PKCα. We developed a high throughput approach by which we identified this compound able to reduce YAP-induced proliferation and clonogenicity of PDAC cellsin vitro, despite it leads to increased YAP nuclear levels. The Hippo pathway is the main inhibitor of YAP. One component of this signaling cascade,Lats1/2, phosphorylates YAP at Serine 127, thereby promoting its cytosolic retention and degradation.In line with YAP nuclear retention after the treatment, we observed that GF 109203X caused an increase of both acetylations and phosphorylations on YAP protein, with the exception of P-S127, suggesting a Hippo pathway-independent mechanism of action of the identified compound. TEAD is the major transcriptional factor partner in the functional activity of YAP and CTGF is considered one of the main representative target genes whose expression is regulated by this couple of transcriptional regulators.Chromatin immunoprecipitation experiments allowed us to demonstrate that GF 109203X interferes with YAP binding to CTGF promoter, without affecting the presence of TEAD at the same region. This inhibition is responsible of CTGF downregulation during GF 109203X administration and it can explain the phenotypic effects we observed. These effects, associated with the observed toxicity in pancreatic cancer cells but not in immortalized HPNE cell lines, make GF 109203X a potential lead compound to be used in drug development for PDAC treatment.
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- 2014
126. Synthesis and preliminary evaluation of Tanshinone Mimic conjugates for mechanism of action studies.
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Assoni G, Assunção Carreira ÁS, Tomiello M, Seneci P, Provenzani A, and Arosio D
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Human antigen R (HuR) is an RNA binding protein (RBP) belonging to the ELAV (Embryonic Lethal Abnormal Vision) family, which stabilizes mRNAs and regulates the expression of multiple genes. Its altered expression or localization is related to pathological features such as cancer or inflammation. Dihydrotanshinone I (DHTS I) is a naturally occurring, tetracyclic ortho-quinone inhibitor of the HuR-mRNA interaction. Our earlier efforts led to the identification of a synthetic Tanshinone Mimic (TM) 2 with improved affinity for HuR. Here we report five new TM probes 3-5 bearing a detection-promoting moiety (either photo affinity probe - PAP or biotin) as a para-substituent on the phenyl-sulphonamide for mechanism of action (MoA) studies. Biological and biochemical assays were used to characterize the novel TM conjugates 3-5. They showed similar toxic activity in HuR-expressing triple-negative breast cancer MDA-MB-231 cells, with micromolar IC50s. REMSAs revealed that photoactivatable groups (4a and 4b), but not biotin (5a and 5b), prevented conjugates' ability to disrupt rHuR-RNA complexes. Further biochemical studies confirmed that biotinylated probes, in particular 5a, can be used to isolate rM1M2 from solutions, taking advantage of streptavidin-coated magnetic beads, thus being the most promising HuR inhibitor to be used for further MoA studies in cell lysates., (© 2024 Wiley‐VCH GmbH.)
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- 2024
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127. Novel, soluble 3-heteroaryl-substituted tanshinone mimics attenuate the inflammatory response in murine macrophages.
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Facen E, Assoni G, Donati G, Paladino D, Carreira A, Bonomo I, Pietra V, Lotti R, Houser J, Fava LL, Seneci P, Marinelli L, Arosio D, and Provenzani A
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- Animals, Mice, Lipopolysaccharides pharmacology, Inflammation drug therapy, ELAV-Like Protein 1 metabolism, Furans pharmacology, Furans chemistry, Humans, Chemokine CXCL10 metabolism, RAW 264.7 Cells, Molecular Dynamics Simulation, Anti-Inflammatory Agents pharmacology, Anti-Inflammatory Agents chemistry, Apoptosis drug effects, Solubility, Abietanes pharmacology, Abietanes chemistry, Macrophages drug effects, Macrophages metabolism
- Abstract
The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR - 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b., (© 2024. The Author(s).)
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- 2024
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128. Pliability in the m 6 A-Binding Region Extends Druggability of YTH Domains.
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Cazzanelli G, Dalle Vedove A, Spagnolli G, Terruzzi L, Colasurdo E, Boldrini A, Patsilinakos A, Sturlese M, Grottesi A, Biasini E, Provenzani A, Quattrone A, and Lolli G
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- Pliability, RNA, Messenger chemistry, RNA, Messenger metabolism, Molecular Conformation, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism
- Abstract
Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m
6 A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6 A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.- Published
- 2024
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129. Interfering with the ERC1-LL5β interaction disrupts plasma membrane-Associated platforms and affects tumor cell motility.
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Ribolla LM, Sala K, Tonoli D, Ramella M, Bracaglia L, Bonomo I, Gonnelli L, Lamarca A, Brindisi M, Pierattelli R, Provenzani A, and de Curtis I
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- Immunoprecipitation, MDA-MB-231 Cells, Humans, Cell Membrane, Cell Movement
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Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5β interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5β and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5β(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5β protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5β(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5β interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5β. Expression of LL5β(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ribolla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2023
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130. Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.
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Carreira ASA, Ravera S, Zucal C, Thongon N, Caffa I, Astigiano C, Bertola N, Buongiorno A, Roccuzzo M, Bisio A, Pardini B, Nencioni A, Bruzzone S, and Provenzani A
- Subjects
- Humans, Cytokines metabolism, Nicotinamide Phosphoribosyltransferase genetics, Nicotinamide Phosphoribosyltransferase metabolism, Mitochondria metabolism, Cell Line, Tumor, Phosphotransferases (Alcohol Group Acceptor), NAD metabolism, Triple Negative Breast Neoplasms drug therapy, Triple Negative Breast Neoplasms genetics
- Abstract
Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD
+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)- Published
- 2023
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131. HuR modulation counteracts lipopolysaccharide response in murine macrophages.
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Bonomo I, Assoni G, La Pietra V, Canarutto G, Facen E, Donati G, Zucal C, Genovese S, Micaelli M, Pérez-Ràfols A, Robbiati S, Kontoyannis DL, De Matteo M, Fragai M, Seneci P, Marinelli L, Arosio D, Piazza S, and Provenzani A
- Subjects
- Mice, Animals, Macrophages metabolism, RNA metabolism, RNA, Messenger genetics, Lipopolysaccharides pharmacology, Lipopolysaccharides metabolism, ELAV-Like Protein 1 genetics, ELAV-Like Protein 1 metabolism
- Abstract
Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)
- Published
- 2023
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132. Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.
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Micaelli M, Dalle Vedove A, Cerofolini L, Vigna J, Sighel D, Zaccara S, Bonomo I, Poulentzas G, Rosatti EF, Cazzanelli G, Alunno L, Belli R, Peroni D, Dassi E, Murakami S, Jaffrey SR, Fragai M, Mancini I, Lolli G, Quattrone A, and Provenzani A
- Abstract
YTHDF proteins bind the N
6 -methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition., Competing Interests: The authors declare the following competing financial interest(s): The authors declare are filed a patent about ebselen analogues. S.R.J. is a scientific advisor and owns stock in 858 Therapeutics., (© 2022 The Authors. Published by American Chemical Society.)- Published
- 2022
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133. HuR-targeted agents: An insight into medicinal chemistry, biophysical, computational studies and pharmacological effects on cancer models.
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Assoni G, La Pietra V, Digilio R, Ciani C, Licata NV, Micaelli M, Facen E, Tomaszewska W, Cerofolini L, Pérez-Ràfols A, Varela Rey M, Fragai M, Woodhoo A, Marinelli L, Arosio D, Bonomo I, Provenzani A, and Seneci P
- Subjects
- Animals, Drug Delivery Systems methods, Gene Silencing, Humans, Inflammation Mediators metabolism, Molecular Weight, Neoplasms drug therapy, RNA, Messenger pharmacology, RNA, Small Interfering pharmacology, ELAV-Like Protein 1 antagonists & inhibitors, ELAV-Like Protein 1 metabolism, Neoplasms physiopathology, RNA metabolism, RNA pharmacology
- Abstract
The Human antigen R (HuR) protein is an RNA-binding protein, ubiquitously expressed in human tissues, that orchestrates target RNA maturation and processing both in the nucleus and in the cytoplasm. A survey of known modulators of the RNA-HuR interactions is followed by a description of its structure and molecular mechanism of action - RRM domains, interactions with RNA, dimerization, binding modes with naturally occurring and synthetic HuR inhibitors. Then, the review focuses on HuR as a validated molecular target in oncology and briefly describes its role in inflammation. Namely, we show ample evidence for the involvement of HuR in the hallmarks and enabling characteristics of cancer, reporting findings from in vitro and in vivo studies; and we provide abundant experimental proofs of a beneficial role for the inhibition of HuR-mRNA interactions through silencing (CRISPR, siRNA) or pharmacological inhibition (small molecule HuR inhibitors)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2022
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134. C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.
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Licata NV, Cristofani R, Salomonsson S, Wilson KM, Kempthorne L, Vaizoglu D, D'Agostino VG, Pollini D, Loffredo R, Pancher M, Adami V, Bellosta P, Ratti A, Viero G, Quattrone A, Isaacs AM, Poletti A, and Provenzani A
- Subjects
- Animals, Cell Line, Codon, Initiator genetics, Cyclic AMP-Dependent Protein Kinases metabolism, DNA Repeat Expansion genetics, Disease Models, Animal, Drosophila drug effects, Frontotemporal Dementia pathology, HEK293 Cells, High-Throughput Screening Assays, Humans, Induced Pluripotent Stem Cells pathology, Isoquinolines pharmacology, Longevity drug effects, Motor Neurons drug effects, Motor Neurons pathology, Protein Biosynthesis drug effects, RNA Interference, Sulfonamides pharmacology, C9orf72 Protein metabolism, Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors, Dipeptides metabolism, Proteolysis drug effects, Small Molecule Libraries pharmacology
- Abstract
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD., (© 2021 The Authors Published under the terms of the CC BY NC ND 4.0 license.)
- Published
- 2022
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135. Hyperinsulinemia and insulin resistance in the obese may develop as part of a homeostatic response to elevated free fatty acids: A mechanistic case-control and a population-based cohort study.
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Fryk E, Olausson J, Mossberg K, Strindberg L, Schmelz M, Brogren H, Gan LM, Piazza S, Provenzani A, Becattini B, Lind L, Solinas G, and Jansson PA
- Subjects
- Adipose Tissue metabolism, Case-Control Studies, Cohort Studies, Diabetes Mellitus, Type 2 complications, Diabetes Mellitus, Type 2 pathology, Fatty Acids, Nonesterified blood, Female, Gene Expression Regulation, Glycerol blood, Glycerol metabolism, Humans, Hyperinsulinism complications, Insulin blood, Insulin Resistance, Lipolysis, Male, Middle Aged, Obesity complications, Principal Component Analysis, Fatty Acids, Nonesterified metabolism, Hyperinsulinism pathology, Obesity pathology
- Abstract
Background: It is commonly accepted that in obesity free fatty acids (FFA) cause insulin resistance and hyperglycemia, which drives hyperinsulinemia. However, hyperinsulinemia is observed in subjects with normoglycaemia and thus the paradigm above should be reevaluated., Methods: We describe two studies: MD-Lipolysis, a case control study investigating the mechanisms of obesity-driven insulin resistance by a systemic metabolic analysis, measurements of adipose tissue lipolysis by microdialysis, and adipose tissue genomics; and POEM, a cohort study used for validating differences in circulating metabolites in relation to adiposity and insulin resistance observed in the MD-Lipolysis study., Findings: In insulin-resistant obese with normal glycaemia from the MD-Lipolysis study, hyperinsulinemia was associated with elevated FFA. Lipolysis, assessed by glycerol release per adipose tissue mass or adipocyte surface, was similar between obese and lean individuals. Adipose tissue from obese subjects showed reduced expression of genes mediating catecholamine-driven lipolysis, lipid storage, and increased expression of genes driving hyperplastic growth. In the POEM study, FFA levels were specifically elevated in obese-overweight subjects with normal fasting glucose and high fasting levels of insulin and C-peptide., Interpretation: In obese subjects with normal glycaemia elevated circulating levels of FFA at fasting are the major metabolic derangement candidate driving fasting hyperinsulinemia. Elevated FFA in obese with normal glycaemia were better explained by increased fat mass rather than by adipose tissue insulin resistance. These results support the idea that hyperinsulinemia and insulin resistance may develop as part of a homeostatic adaptive response to increased adiposity and FFA., Funding: Swedish-Research-Council (2016-02660); Diabetesfonden (DIA2017-250; DIA2018-384; DIA2020-564); Novo-Nordisk-Foundation (NNF17OC0027458; NNF19OC0057174); Cancerfonden (CAN2017/472; 200840PjF); Swedish-ALF-agreement (2018-74560)., Competing Interests: Declaration of Competing Interest L.M.G holds an employment at AstraZeneca R&D. All other authors declare no competing interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
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136. Multilayer and MATR3-dependent regulation of mRNAs maintains pluripotency in human induced pluripotent stem cells.
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Pollini D, Loffredo R, Maniscalco F, Cardano M, Micaelli M, Bonomo I, Licata NV, Peroni D, Tomaszewska W, Rossi A, Crippa V, Dassi E, Viero G, Quattrone A, Poletti A, Conti L, and Provenzani A
- Abstract
Matrin3 (MATR3) is a nuclear RNA/DNA-binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells, and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced pluripotent stem cells (hiPSCs). MATR3 downregulation affects hiPSCs' differentiation potential by altering key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts., Competing Interests: The authors declare no competing interest., (© 2021 The Author(s).)
- Published
- 2021
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137. Author Correction: Fasting-mimicking diet and hormone therapy induce breast cancer regression.
- Author
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Caffa I, Spagnolo V, Vernieri C, Valdemarin F, Becherini P, Wei M, Brandhorst S, Zucal C, Driehuis E, Ferrando L, Piacente F, Tagliafico A, Cilli M, Mastracci L, Vellone VG, Piazza S, Cremonini AL, Gradaschi R, Mantero C, Passalacqua M, Ballestrero A, Zoppoli G, Cea M, Arrighi A, Odetti P, Monacelli F, Salvadori G, Cortellino S, Clevers H, De Braud F, Sukkar SG, Provenzani A, Longo VD, and Nencioni A
- Published
- 2020
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138. Fasting-mimicking diet and hormone therapy induce breast cancer regression.
- Author
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Caffa I, Spagnolo V, Vernieri C, Valdemarin F, Becherini P, Wei M, Brandhorst S, Zucal C, Driehuis E, Ferrando L, Piacente F, Tagliafico A, Cilli M, Mastracci L, Vellone VG, Piazza S, Cremonini AL, Gradaschi R, Mantero C, Passalacqua M, Ballestrero A, Zoppoli G, Cea M, Arrighi A, Odetti P, Monacelli F, Salvadori G, Cortellino S, Clevers H, De Braud F, Sukkar SG, Provenzani A, Longo VD, and Nencioni A
- Subjects
- Animals, Biological Factors blood, Breast Neoplasms pathology, Diet, Healthy methods, Disease Models, Animal, Disease Progression, Drug Resistance, Neoplasm drug effects, Early Growth Response Protein 1 metabolism, Female, Fulvestrant administration & dosage, Humans, Insulin blood, Insulin-Like Growth Factor I metabolism, Leptin blood, MCF-7 Cells, Mice, Inbred NOD, Mice, SCID, PTEN Phosphohydrolase metabolism, Piperazines administration & dosage, Piperazines therapeutic use, Pyridines administration & dosage, Pyridines therapeutic use, Receptors, Estrogen, Receptors, Progesterone, Tamoxifen adverse effects, Tamoxifen therapeutic use, Xenograft Model Antitumor Assays, Breast Neoplasms diet therapy, Breast Neoplasms drug therapy, Diet Therapy methods, Fasting physiology, Fulvestrant therapeutic use
- Abstract
Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit
1,2 . Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.- Published
- 2020
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139. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.
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Notarangelo M, Zucal C, Modelska A, Pesce I, Scarduelli G, Potrich C, Lunelli L, Pederzolli C, Pavan P, la Marca G, Pasini L, Ulivi P, Beltran H, Demichelis F, Provenzani A, Quattrone A, and D'Agostino VG
- Subjects
- Case-Control Studies, Cell Line, Tumor, Flow Cytometry, Humans, Liquid Biopsy standards, Neoplasms genetics, Neoplasms metabolism, Polymerase Chain Reaction, Sensitivity and Specificity, Ultracentrifugation, Biomarkers, Tumor blood, Extracellular Vesicles metabolism, Extracellular Vesicles ultrastructure, Liquid Biopsy methods, Neoplasms blood, Neoplasms diagnosis, Nickel
- Abstract
Background: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice., Methods: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility., Findings: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 10
10 /ml) and large EV (~560 nm of mean diameter; ~5 × 108 /ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies., Interpretation: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur)., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2019
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140. Screening Approaches for Targeting Ribonucleoprotein Complexes: A New Dimension for Drug Discovery.
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D'Agostino VG, Sighel D, Zucal C, Bonomo I, Micaelli M, Lolli G, Provenzani A, Quattrone A, and Adami V
- Subjects
- Drug Delivery Systems, Protein Binding, RNA-Binding Proteins metabolism, Ribonucleoproteins metabolism, Untranslated Regions, Drug Discovery methods, High-Throughput Screening Assays methods, Ribonucleoproteins drug effects
- Abstract
RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts.
- Published
- 2019
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141. Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38 MAPK , and JNK Kinase Pathways.
- Author
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Marchesi N, Thongon N, Pascale A, Provenzani A, Koskela A, Korhonen E, Smedowski A, Govoni S, Kauppinen A, Kaarniranta K, and Amadio M
- Subjects
- Autophagy physiology, Cell Line, Humans, MAP Kinase Kinase 4 metabolism, Retinal Pigment Epithelium cytology, Retinal Pigment Epithelium enzymology, p38 Mitogen-Activated Protein Kinases metabolism, ELAV-Like Protein 1 metabolism, MAP Kinase Signaling System, Retinal Pigment Epithelium metabolism, Sequestosome-1 Protein metabolism
- Abstract
RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38
MAPK , PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.- Published
- 2018
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142. The CDKN2A/p16(INK) (4a) 5'UTR sequence and translational regulation: impact of novel variants predisposing to melanoma.
- Author
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Andreotti V, Bisio A, Bressac-de Paillerets B, Harland M, Cabaret O, Newton-Bishop J, Pastorino L, Bruno W, Bertorelli R, De Sanctis V, Provenzani A, Menin C, Fronza G, Queirolo P, Spitale RC, Bianchi-Scarrà G, Inga A, and Ghiorzo P
- Subjects
- Cyclin-Dependent Kinase Inhibitor p16 biosynthesis, Female, Humans, Male, Melanoma metabolism, Pedigree, 5' Untranslated Regions, Cyclin-Dependent Kinase Inhibitor p16 genetics, Genetic Predisposition to Disease, Genetic Variation, Melanoma genetics, Peptide Chain Initiation, Translational genetics
- Abstract
Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5'UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.-27del23, c.-93-91delAGG) were classified as causal mutations (score ≥3), along with the c.-21C>T, c.-34G>T, and c.-56G>T, which had already been studied by a subset of assays. The novel c.-42T>A as well as the previously described c.-67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5'UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16(INK) (4a) translation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2016
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143. Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells.
- Author
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Latorre E, Carelli S, Caremoli F, Giallongo T, Colli M, Canazza A, Provenzani A, Di Giulio AM, and Gorio A
- Subjects
- Cells, Cultured, Female, Humans, Male, Protein Binding physiology, ELAV-Like Protein 1 metabolism, Mesenchymal Stem Cells metabolism, RNA, Messenger physiology, SOXB1 Transcription Factors physiology
- Abstract
Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2016
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144. The 5'-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding.
- Author
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Bisio A, Latorre E, Andreotti V, Bressac-de Paillerets B, Harland M, Scarra GB, Ghiorzo P, Spitale RC, Provenzani A, and Inga A
- Subjects
- Blotting, Western, Cell Hypoxia, Cell Line, Tumor, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Gene Expression Regulation, Neoplastic drug effects, HCT116 Cells, Humans, MCF-7 Cells, Melanoma genetics, Melanoma metabolism, Melanoma pathology, Mutation, Naphthyridines pharmacology, Protein Binding, RNA Interference, Sirolimus pharmacology, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases metabolism, Y-Box-Binding Protein 1 metabolism, 5' Untranslated Regions genetics, Cyclin-Dependent Kinase Inhibitor p16 genetics, Internal Ribosome Entry Sites genetics, Protein Biosynthesis, RNA, Messenger genetics, Y-Box-Binding Protein 1 genetics
- Abstract
CDKN2A/p16INK4a is an essential tumor suppressor gene that controls cell cycle progression and replicative senescence. It is also the main melanoma susceptibility gene. Here we report that p16INK4a 5'UTR mRNA acts as a cellular Internal Ribosome Entry Site (IRES). The potential for p16INK4a 5'UTR to drive cap-independent translation was evaluated by dual-luciferase assays using bicistronic and monocistronic vectors. Results of reporters' relative activities coupled to control analyses for actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5'UTR could stimulate cap-independent translation. Notably, hypoxic stress and the treatment with mTOR inhibitors enhanced the translation-stimulating property of p16INK4a 5'UTR. RNA immunoprecipitation performed in melanoma-derived SK-Mel-28 and in a patient-derived lymphoblastoid cell line indicated that YBX1 can bind the wild type p16INK4a mRNA increasing its translation efficiency, particularly during hypoxic stress. Modulation of YBX1 expression further supported its involvement in cap-independent translation of the wild type p16INK4a but not a c.-42T>A variant. RNA SHAPE assays revealed local flexibility changes for the c.-42T>A variant at the predicted YBX1 binding site region. Our results indicate that p16INK4a 5'UTR contains a cellular IRES that can enhance mRNA translation efficiency, in part through YBX1.
- Published
- 2015
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145. [Immuknow and long term kidney graft].
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Andreotti C, Zortea M, Provenzani A, Gentilini M, and Bucella N
- Subjects
- CD4 Antigens blood, Female, Humans, Immunity, Cellular, Male, Middle Aged, Skin Neoplasms complications, Time Factors, Graft Survival immunology, Immunologic Tests, Immunosuppressive Agents therapeutic use, Kidney Transplantation
- Abstract
Introduction: The survival of transplanted kidneys has improved over time, but there is an increased risk of neoplastic disease. In the long time follow up, non-melanoma skin cancers (NMSC) are the most frequent diseases and at the time of the occurrence of a NMSC we should evaluate a reduction or a change of IS. From a clinical point of view, the evaluation of immunosuppression is still a problem. The Immuknow assay may be of help in evaluating the immune response of transplanted patients. Here, by means of the ImmKnow assay, we tried to evaluate if long term renal transplant patients with NMSC are more immunosuppressed than patients without NMSC., Methods: 33 long term kidney transplant patients, 16 with NMSC and 17 without NMSC, were recruited and blood samples were drawn at baseline, 4 months, 8 months and 12 months to check renal function, blood levels of cni and to perform immuknow assay., Results: most values of T CD4+ reactivity were comprised between (atp) 225 and 525 ng/ml as for an moderate immunosuppression. No major differences have been observed between the two groups. No correlation with blood level of CNI was detected. T CD4+ activity changed over time for both the groups. 3 patients of the group without NMSC had levels of CD4+ reactivity constantly under (ATP) 225 ng/ml, classified as low per manifacturers definition., Conclusion: in our limited experience the measure of cell-mediated immunity by immuknow assay years after transplantation, has not evidenced any significant difference between patients positive for NMSC and negative patients. We observed variation of the CD4 reactivity with time, no correlation with the level of CNI and the useful identification of some cases of low levels of cell reactivity.
- Published
- 2015
146. A novel high throughput biochemical assay to evaluate the HuR protein-RNA complex formation.
- Author
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D'Agostino VG, Adami V, and Provenzani A
- Subjects
- AU Rich Elements, Binding, Competitive, Cell Line, Humans, Kinetics, Oligoribonucleotides metabolism, Protein Binding, Protein Transport, RNA, Messenger chemistry, Recombinant Proteins metabolism, Tristetraprolin metabolism, ELAV Proteins metabolism, High-Throughput Screening Assays methods, RNA, Messenger metabolism
- Abstract
The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize in vitro its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3' untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated Kd of 2.5±0.60 nM, k on of 2.76±0.56*10(6) M(-1) min(-1), and k off of 0.007±0.005 min(-1). We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity.
- Published
- 2013
- Full Text
- View/download PDF
147. mRNA fate: Life and death of the mRNA in the cytoplasm.
- Author
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Denti MA, Viero G, Provenzani A, Quattrone A, and Macchi P
- Subjects
- Animals, Gene Expression Regulation, Humans, MicroRNAs chemistry, MicroRNAs genetics, MicroRNAs metabolism, Phenotype, Protein Biosynthesis, Proteins chemistry, Proteins metabolism, Proteome, RNA Stability, RNA, Messenger chemistry, Cytoplasm metabolism, RNA, Messenger genetics, RNA, Messenger metabolism
- Abstract
The life of an mRNA molecule begins with transcription and ultimately ends in degradation. In the course of its life, however, mRNA is examined, modified in various ways and transported before eventually being translated into proteins. All these processes are performed by proteins and non-coding RNAs whose complex interplay in the cell contributes to determining the proteome changes and the phenotype of cells. On May 23‒26, 2012, over 150 scientists from around the world convened in the sunny shores of Riva del Garda, Italy, for the workshop entitled: "mRNA fate: Life and Death of mRNA in the Cytoplasm." Sessions included mRNA trafficking, mRNA translational control, RNA metabolism and disease, RNA-protein structures and systems biology of RNA. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research.
- Published
- 2013
- Full Text
- View/download PDF
148. Experimentally exploring the conformational space sampled by domain reorientation in calmodulin.
- Author
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Bertini I, Del Bianco C, Gelis I, Katsaros N, Luchinat C, Parigi G, Peana M, Provenzani A, and Zoroddu MA
- Subjects
- Animals, Anisotropy, Calmodulin genetics, Calmodulin metabolism, Humans, Protein Conformation, Protein Structure, Tertiary, Calmodulin chemistry
- Abstract
The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb(3+)- and Tm(3+)-substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by approximately 30 degrees with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither the extended helix conformation initially observed in the solid state for free calcium calmodulin nor the closed conformation(s) adopted by calcium calmodulin either alone or in its adduct(s) with target peptide(s) is among the most preferred ones. These findings are unique, both in terms of structural information obtained on a biomolecule that samples multiple conformations and in terms of the approach developed to achieve the results. The same approach is in principle applicable to other multidomain proteins, as well as to multiple interaction modes between two macromolecular partners.
- Published
- 2004
- Full Text
- View/download PDF
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