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Article ; Online: Pliability in the m

Cazzanelli, Giulia / Dalle Vedove, Andrea / Spagnolli, Giovanni / Terruzzi, Luca / Colasurdo, Enrica / Boldrini, Alberto / Patsilinakos, Alexandros / Sturlese, Mattia / Grottesi, Alessandro / Biasini, Emiliano / Provenzani, Alessandro / Quattrone, Alessandro / Lolli, Graziano

Journal of chemical information and modeling

2024 Volume 64, Issue 5, Page(s) 1682–1690

Abstract: Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the ... ...

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
MeSH term(s)	RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; Pliability ; RNA, Messenger/chemistry ; RNA, Messenger/metabolism ; Molecular Conformation
Chemical Substances	RNA-Binding Proteins ; RNA, Messenger
Language	English
Publishing date	2024-02-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	190019-5
ISSN	1549-960X ; 0095-2338
ISSN (online)	1549-960X
ISSN	0095-2338
DOI	10.1021/acs.jcim.4c00051
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Article ; Online: Interfering with the ERC1-LL5β interaction disrupts plasma membrane-Associated platforms and affects tumor cell motility.

Ribolla, Lucrezia Maria / Sala, Kristyna / Tonoli, Diletta / Ramella, Martina / Bracaglia, Lorenzo / Bonomo, Isabelle / Gonnelli, Leonardo / Lamarca, Andrea / Brindisi, Matteo / Pierattelli, Roberta / Provenzani, Alessandro / de Curtis, Ivan

PloS one

2023 Volume 18, Issue 7, Page(s) e0287670

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Cell Membrane ; Cell Movement ; Immunoprecipitation ; MDA-MB-231 Cells ; Humans
Chemical Substances	ERC1 protein, human ; PHLDB2 protein, human
Language	English
Publishing date	2023-07-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0287670
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Article ; Online: Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.

Carreira, Agata Sofia Assuncao / Ravera, Silvia / Zucal, Chiara / Thongon, Natthakan / Caffa, Irene / Astigiano, Cecilia / Bertola, Nadia / Buongiorno, Arianna / Roccuzzo, Michela / Bisio, Alessandra / Pardini, Barbara / Nencioni, Alessio / Bruzzone, Santina / Provenzani, Alessandro

Neoplasia (New York, N.Y.)

2023 Volume 41, Page(s) 100903

Abstract: Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in ... ...

Abstract	Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD
MeSH term(s)	Humans ; NAD/metabolism ; Triple Negative Breast Neoplasms/drug therapy ; Triple Negative Breast Neoplasms/genetics ; Cytokines/metabolism ; Nicotinamide Phosphoribosyltransferase/genetics ; Nicotinamide Phosphoribosyltransferase/metabolism ; Mitochondria/metabolism ; Cell Line, Tumor ; Phosphotransferases (Alcohol Group Acceptor)
Chemical Substances	NAD (0U46U6E8UK) ; Cytokines ; Nicotinamide Phosphoribosyltransferase (EC 2.4.2.12) ; NMRK1 protein, human (EC 2.7.1.22) ; Phosphotransferases (Alcohol Group Acceptor) (EC 2.7.1.-)
Language	English
Publishing date	2023-05-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1483840-0
ISSN	1476-5586 ; 1522-8002
ISSN (online)	1476-5586
ISSN	1522-8002
DOI	10.1016/j.neo.2023.100903
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Article ; Online: HuR modulation counteracts lipopolysaccharide response in murine macrophages.

Bonomo, Isabelle / Assoni, Giulia / La Pietra, Valeria / Canarutto, Giulia / Facen, Elisa / Donati, Greta / Zucal, Chiara / Genovese, Silvia / Micaelli, Mariachiara / Pérez-Ràfols, Anna / Robbiati, Sergio / Kontoyannis, Dimitris L / De Matteo, Marilenia / Fragai, Marco / Seneci, Pierfausto / Marinelli, Luciana / Arosio, Daniela / Piazza, Silvano / Provenzani, Alessandro

Disease models & mechanisms

2023 Volume 16, Issue 3

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 ... ...

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.
MeSH term(s)	Mice ; Animals ; Lipopolysaccharides/pharmacology ; Lipopolysaccharides/metabolism ; ELAV-Like Protein 1/genetics ; ELAV-Like Protein 1/metabolism ; Macrophages/metabolism ; RNA/metabolism ; RNA, Messenger/genetics
Chemical Substances	Lipopolysaccharides ; ELAV-Like Protein 1 ; RNA (63231-63-0) ; RNA, Messenger
Language	English
Publishing date	2023-03-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2451104-3
ISSN	1754-8411 ; 1754-8403
ISSN (online)	1754-8411
ISSN	1754-8403
DOI	10.1242/dmm.050120
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Article ; Online: Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids.

Notarangelo, Michela / Ferrara, Deborah / Potrich, Cristina / Lunelli, Lorenzo / Vanzetti, Lia / Provenzani, Alessandro / Basso, Manuela / Quattrone, Alessandro / D'Agostino, Vito G

Bio-protocol

2020 Volume 10, Issue 3, Page(s) e3512

Abstract: Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies ... ...

Abstract	Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.
Language	English
Publishing date	2020-02-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325 ; 2331-8325
ISSN (online)	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.3512
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Article ; Online: A novel high throughput biochemical assay to evaluate the HuR protein-RNA complex formation.

D'Agostino, Vito G / Adami, Valentina / Provenzani, Alessandro

PloS one

2013 Volume 8, Issue 8, Page(s) e72426

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, ... ...

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.
MeSH term(s)	AU Rich Elements ; Binding, Competitive ; Cell Line ; ELAV Proteins/metabolism ; High-Throughput Screening Assays/methods ; Humans ; Kinetics ; Oligoribonucleotides/metabolism ; Protein Binding ; Protein Transport ; RNA, Messenger/chemistry ; RNA, Messenger/metabolism ; Recombinant Proteins/metabolism ; Tristetraprolin/metabolism
Chemical Substances	ELAV Proteins ; Oligoribonucleotides ; RNA, Messenger ; Recombinant Proteins ; Tristetraprolin ; ZFP36 protein, human
Language	English
Publishing date	2013-08-12
Publishing country	United States
Document type	Journal Article
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0072426
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Article ; Online: Identification and Characterization of an RRM-Containing, RNA Binding Protein in

Ciani, Caterina / Pérez-Ràfols, Anna / Bonomo, Isabelle / Micaelli, Mariachiara / Esposito, Alfonso / Zucal, Chiara / Belli, Romina / D'Agostino, Vito Giuseppe / Bianconi, Irene / Calderone, Vito / Cerofolini, Linda / Massidda, Orietta / Whalen, Michael Bernard / Fragai, Marco / Provenzani, Alessandro

Biomolecules

2022 Volume 12, Issue 7

Abstract: Acinetobacter ... ...

Abstract	Acinetobacter baumannii
MeSH term(s)	Acinetobacter baumannii/genetics ; Acinetobacter baumannii/metabolism ; Animals ; Carrier Proteins/metabolism ; Humans ; Protein Binding/genetics ; Proteome/metabolism ; RNA/metabolism ; RNA Recognition Motif/genetics ; RNA-Binding Proteins/metabolism
Chemical Substances	Carrier Proteins ; Proteome ; RNA-Binding Proteins ; RNA (63231-63-0)
Language	English
Publishing date	2022-06-30
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2701262-1
ISSN	2218-273X ; 2218-273X
ISSN (online)	2218-273X
ISSN	2218-273X
DOI	10.3390/biom12070922
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Article ; Online: 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.

Fryk, Emanuel / Olausson, Josefin / Mossberg, Karin / Strindberg, Lena / Schmelz, Martin / Brogren, Helén / Gan, Li-Ming / Piazza, Silvano / Provenzani, Alessandro / Becattini, Barbara / Lind, Lars / Solinas, Giovanni / Jansson, Per-Anders

EBioMedicine

2021 Volume 65, Page(s) 103264

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 ... ...

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).
MeSH term(s)	Adipose Tissue/metabolism ; Case-Control Studies ; Cohort Studies ; Diabetes Mellitus, Type 2/complications ; Diabetes Mellitus, Type 2/pathology ; Fatty Acids, Nonesterified/blood ; Fatty Acids, Nonesterified/metabolism ; Female ; Gene Expression Regulation ; Glycerol/blood ; Glycerol/metabolism ; Humans ; Hyperinsulinism/complications ; Hyperinsulinism/pathology ; Insulin/blood ; Insulin Resistance ; Lipolysis ; Male ; Middle Aged ; Obesity/complications ; Obesity/pathology ; Principal Component Analysis
Chemical Substances	Fatty Acids, Nonesterified ; Insulin ; Glycerol (PDC6A3C0OX)
Language	English
Publishing date	2021-03-09
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2851331-9
ISSN	2352-3964
ISSN (online)	2352-3964
DOI	10.1016/j.ebiom.2021.103264
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Zs.A 7090: Show issues

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Article ; Online: Screening Approaches for Targeting Ribonucleoprotein Complexes: A New Dimension for Drug Discovery.

D'Agostino, Vito Giuseppe / Sighel, Denise / Zucal, Chiara / Bonomo, Isabelle / Micaelli, Mariachiara / Lolli, Graziano / Provenzani, Alessandro / Quattrone, Alessandro / Adami, Valentina

SLAS discovery : advancing life sciences R & D

2019 Volume 24, Issue 3, Page(s) 314–331

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 ... ...

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.
MeSH term(s)	Drug Delivery Systems ; Drug Discovery/methods ; High-Throughput Screening Assays/methods ; Protein Binding ; RNA-Binding Proteins/metabolism ; Ribonucleoproteins/drug effects ; Ribonucleoproteins/metabolism ; Untranslated Regions
Chemical Substances	RNA-Binding Proteins ; Ribonucleoproteins ; Untranslated Regions
Language	English
Publishing date	2019-01-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1177/2472555218818065
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Zs.A 4911: Show issues

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Article ; Online: Valutazione della immunità cellulo mediata in pazienti che hanno e che non hanno sviluppato tumori cutanei non melanoma (NMSC) nel follow up a lungo termine del trapianto renale.

Andreotti, Cristina / Zortea, Michela / Provenzani, Alessandro / Gentilini, Maria / Bucella, Nadia

Giornale italiano di nefrologia : organo ufficiale della Societa italiana di nefrologia

2015 Volume 32, Issue 2

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 ... ...

Title translation	Immuknow and long term kidney graft.
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.
MeSH term(s)	CD4 Antigens/blood ; Female ; Graft Survival/immunology ; Humans ; Immunity, Cellular ; Immunologic Tests ; Immunosuppressive Agents/therapeutic use ; Kidney Transplantation ; Male ; Middle Aged ; Skin Neoplasms/complications ; Time Factors
Chemical Substances	CD4 Antigens ; Immunosuppressive Agents
Language	Italian
Publishing date	2015-03
Publishing country	Italy
Document type	Journal Article
ZDB-ID	1237110-5
ISSN	1724-5990 ; 0393-5590
ISSN (online)	1724-5990
ISSN	0393-5590
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