Papers by Alessandro Provenzani
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in... more 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 conforma-tion 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 mR-NAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without sys-temic toxicity.
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[Immuknow and long term kidney graft]
Giornale italiano di nefrologia : organo ufficiale della Società italiana di nefrologia
The survival of transplanted kidneys has improved over time, but there is an increased risk of ne... more 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. 33 long term kidney transplant patients, 16 with NMSCand 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. most values of T CD4+ reactivity were comprised between (atp) 225 and 525 ng/ml as for an moderate immunosuppressi...
Human antigen R binding and regulation of SOX2 mRNA in human mesenchymal stem cells
Molecular Pharmacology, 2015
Since 2005, SOX2 has raised the attention of the scientific community for being one of the key tr... more Since 2005, SOX2 has raised 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 acting on nucleic acids primarily used to treat certain types of cancer, and recently re-addressed to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. Additionally, mitoxantrone has been shown to inhibit the binding of Human antigen R (HuR) RNA binding protein to TNF alpha mRNA. Our results show that HuR binds to the 3'UTR region of SOX2 mRNA together with the RNA-induced silencing complex-miR145. The HuR binding works by stabilizing the interaction between the 3' UTR and RISC. Cell exposure to mitoxantrone leads to HuR detachment, and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows to an 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.
The GSK3β inhibitor BIS I reverts YAP-dependent EMT signature in PDAC cell lines by decreasing SMADs expression level
Oncotarget, 2014
Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases
Scientific Reports, 2016
Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein incl... more Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.
Glucosylation of Isatin-3-Oxime followed by 2Din situ NMR in Plant Cells at Highest Magnetic Field without Labelling
Http Dx Doi Org 10 1080 10575630108041268, Oct 4, 2006
The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gra... more 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.
Bioorganic Medicinal Chemistry, Sep 1, 2003
Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic h... more Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne were carried out. When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13 C. Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800). Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe TM that the alkaloid enters two metabolic routes. Vinorine is intracellularly transformed on route I through vellosimine and 10-deoxysarpagine into sarpagine. On route II, the alkaloid is converted by hydroxylation through vomilenine into the glucoside raucaffricine. Intracellular alkaloid concentrations of $ 500 mM are measurable in vivo with cryogenic NMR technology. # Abbreviations: HSQC, heteronuclear single-quantum coherence; HMQC, heteronuclear multiple-quantum coherence.
The 5'-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding
Oncotarget, Jan 17, 2015
CDKN2A/p16INK4a is an essential tumor suppressor gene that controls cell cycle progression and re... more 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 duri...
The CDKN2A/p16(INK) (4a) 5'UTR sequence and translational regulation: impact of novel variants predisposing to melanoma
Pigment cell & melanoma research, Jan 18, 2015
Many variants of uncertain functional significance in cancer susceptibility genes lie in regulato... more 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 (9 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...
Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function
Scientific Reports, 2015
Post-transcriptional regulation is an essential determinant of gene expression programs in physio... more 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.
EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition
BMC Cancer, 2015
Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis f... more Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.
Current Drug Targets, 2015
The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic ... more 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.
PLoS ONE, 2013
The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization a... more 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. Citation: D'Agostino VG, Adami V, Provenzani A (2013) A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation. PLoS ONE 8(8): e72426.
Per2 plays a major role in the control of liver glycogen metabolism and fasting glycaemia
PER2 promotes glucose storage to liver glycogen during feeding and acute fasting by inducing Gys2 PTG and GL expression
Molecular Metabolism, 2013
The interplay between hepatic glycogen metabolism and blood glucose levels is a paradigm of the r... more 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. Per2 (Brdm1) mice show reduced hepatic glycogen content and altered circadian expression during controlled fasting and refeeding. Livers from Per2 (Brdm1) 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 G L . 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.
Nucleic Acids Research, 2013
Little is known regarding the post-transcriptional networks that control gene expression in eukar...
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Papers by Alessandro Provenzani