Publications by authors named "Claudio Santoro"

57 Publications

SINEUPs: a novel toolbox for RNA therapeutics.

Essays Biochem 2021 Oct 8. Epub 2021 Oct 8.

Central RNA Laboratory, Istituto Italiano di Tecnologia (IIT), Genova, Italy.

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.
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http://dx.doi.org/10.1042/EBC20200114DOI Listing
October 2021

Gut-Ex-Vivo system as a model to study gluten response in celiac disease.

Cell Death Discov 2021 Mar 12;7(1):45. Epub 2021 Mar 12.

Department of Health Science, University of Piemonte Orientale, Novara, Italy.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.
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http://dx.doi.org/10.1038/s41420-021-00430-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955131PMC
March 2021

Novel Bispecific Antibody for Synovial-Specific Target Delivery of Anti-TNF Therapy in Rheumatoid Arthritis.

Front Immunol 2021 19;12:640070. Epub 2021 Feb 19.

Department of Experimental Medicine and Rheumatology, Barts and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary University of London, London, United Kingdom.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.
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http://dx.doi.org/10.3389/fimmu.2021.640070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933454PMC
September 2021

Systemic Cat-Scratch Disease: a "Troublesome" Diagnosis.

Pediatr Infect Dis J 2021 03;40(3):e117-e119

Department of Pediatrics, Santobono-Pausilipon Children's Hospital, Naples, Italy.

Diagnosis of systemic cat scratch disease may be challenging. Here, we describe a case of an immunocompetent girl exhibiting fever and multifocal hepatosplenic abscesses. Diagnostic tests for Bartonella henselae infection (enzyme immunoassay and polymerase chain reaction) were found steadily negative and the diagnosis, suspected on the basis of the Margilet's criteria, was finally confirmed by indirect immunofluorescent antibodies.
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http://dx.doi.org/10.1097/INF.0000000000002996DOI Listing
March 2021

Defining the Disease-Specific Antigenic Repertoire.

Front Microbiol 2020 9;11:1551. Epub 2020 Jul 9.

Institute of Genetic and Biomedical Research, UoS Milan, National Research Council, Milan, Italy.

The analysis of the interaction between (HP) and the host is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific "disease signature" with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines "phage display" and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen's genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future.
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http://dx.doi.org/10.3389/fmicb.2020.01551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396715PMC
July 2020

Scurvy in childhood: don't forget it.

Minerva Pediatr 2020 Jul 7. Epub 2020 Jul 7.

Department of Paediatrics AORN "Santobono-Pausilipon", Naples, Italy.

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http://dx.doi.org/10.23736/S0026-4946.20.05586-3DOI Listing
July 2020

InteractomeSeq: a web server for the identification and profiling of domains and epitopes from phage display and next generation sequencing data.

Nucleic Acids Res 2020 07;48(W1):W200-W207

Institute for Biomedical Technologies, National Research Council, Bari 70100, Italy.

High-Throughput Sequencing technologies are transforming many research fields, including the analysis of phage display libraries. The phage display technology coupled with deep sequencing was introduced more than a decade ago and holds the potential to circumvent the traditional laborious picking and testing of individual phage rescued clones. However, from a bioinformatics point of view, the analysis of this kind of data was always performed by adapting tools designed for other purposes, thus not considering the noise background typical of the 'interactome sequencing' approach and the heterogeneity of the data. InteractomeSeq is a web server allowing data analysis of protein domains ('domainome') or epitopes ('epitome') from either Eukaryotic or Prokaryotic genomic phage libraries generated and selected by following an Interactome sequencing approach. InteractomeSeq allows users to upload raw sequencing data and to obtain an accurate characterization of domainome/epitome profiles after setting the parameters required to tune the analysis. The release of this tool is relevant for the scientific and clinical community, because InteractomeSeq will fill an existing gap in the field of large-scale biomarkers profiling, reverse vaccinology, and structural/functional studies, thus contributing essential information for gene annotation or antigen identification. InteractomeSeq is freely available at https://InteractomeSeq.ba.itb.cnr.it/.
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http://dx.doi.org/10.1093/nar/gkaa363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319578PMC
July 2020

Aldo-keto reductases protect metastatic melanoma from ER stress-independent ferroptosis.

Cell Death Dis 2019 11 28;10(12):902. Epub 2019 Nov 28.

Department of Health Sciences, University of Piemonte Orientale, Novara, Italy.

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.
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http://dx.doi.org/10.1038/s41419-019-2143-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883066PMC
November 2019

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia.

Nucleic Acids Res 2019 11;47(20):10728-10743

Central RNA Laboratory, Istituto Italiano di Tecnologia (IIT), Genova, Italy.

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.
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http://dx.doi.org/10.1093/nar/gkz798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6847766PMC
November 2019

The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.

FASEB J 2019 12 25;33(12):13572-13589. Epub 2019 Oct 25.

Area of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy.

Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C-terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)-enhancing translation of sense protein-coding mRNAs. By using RNA-interacting domainome technology, we identify the IL enhancer-binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA-binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements-enhanced cross-linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome-wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA-binding protein, strengthening the model of TEs as functional modules in lncRNAs.-Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.
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http://dx.doi.org/10.1096/fj.201901618RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894054PMC
December 2019

SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease.

Mol Ther 2020 02 16;28(2):642-652. Epub 2019 Aug 16.

Central RNA Laboratory and Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia (IIT), 16263 Genova, Italy. Electronic address:

Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD.
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http://dx.doi.org/10.1016/j.ymthe.2019.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000958PMC
February 2020

High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

Oncoimmunology 2019;8(9):e1614856. Epub 2019 Jun 4.

Department of Life Science, University of Trieste, Trieste, Italy.

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.
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http://dx.doi.org/10.1080/2162402X.2019.1614856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685609PMC
February 2021

Antisense Transcription in Loci Associated to Hereditary Neurodegenerative Diseases.

Mol Neurobiol 2019 Aug 4;56(8):5392-5415. Epub 2019 Jan 4.

Area of Neuroscience, SISSA, Trieste, Italy.

Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.
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http://dx.doi.org/10.1007/s12035-018-1465-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614138PMC
August 2019

Author Correction: Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

Sci Rep 2018 Nov 16;8(1):17227. Epub 2018 Nov 16.

Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-35522-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237841PMC
November 2018

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing.

J Vis Exp 2018 10 3(140). Epub 2018 Oct 3.

Institute of Genetic and Biomedical Research, National Research Council, Rozzano, Milan, Italy; Humanitas Clinical and Research Center, Rozzano, Milan, Italy;

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 β-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.
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http://dx.doi.org/10.3791/56981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235410PMC
October 2018

Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.

FASEB J 2019 02 4;33(2):2327-2342. Epub 2018 Oct 4.

Department of Life Sciences, University of Trieste, Trieste, Italy.

The interaction between the enzyme transglutaminase 2 (TG2) and fibronectin (FN) is involved in the cell-matrix interactions that regulate cell signaling, adhesion, and migration and play central roles in pathologic conditions, particularly fibrosis and cancer. A precise definition of the exact interaction domains on both proteins could provide a tool to design novel molecules with potential therapeutic applications. Although specific residues involved in the interaction within TG2 have been analyzed, little is known regarding the TG2 binding site on FN. This site has been mapped to a large internal 45-kDa protein fragment coincident with the gelatin binding domain (GBD). With the goal of defining the minimal FN interacting domain for TG2, we produced several expression constructs encoding different portions or modules of the GBD and tested their binding and functional properties. The results demonstrate that the I module is necessary and sufficient for TG2-binding in vitro, but does not have functional effects on TG2-expressing cells. Modules I and I increase the strength of the binding and are required for cell adhesion. A 15-kDa fragment encompassing modules I behaves as the whole 45-kDa GBD and mediates signaling, adhesion, spreading, and migration of TG2 cells. This study provides new insights into the mechanism for TG2 binding to FN.-Soluri, M. F., Boccafoschi, F., Cotella, D., Moro, L., Forestieri, G., Autiero, I., Cavallo, L., Oliva, R., Griffin, M., Wang, Z., Santoro, C., Sblattero, D. Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.
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http://dx.doi.org/10.1096/fj.201800054RRRDOI Listing
February 2019

Ecto-Calreticulin is essential for an efficient immunogenic cell death stimulation in mouse melanoma.

Genes Immun 2019 07 4;20(6):509-513. Epub 2018 Oct 4.

Department of Health Science (DISS), University of 'Piemonte Orientale', Novara, Italy.

Skin melanoma remains one of the most aggressive and difficult to treat human malignancy, with an increasing incidence every year. Although surgical resection represents the best therapeutic approach, this is only feasible in cases of early diagnosis. Furthermore, the established malignancy is resistant to all therapeutic strategies employed so far, resulting in an unacceptable patient survival rate. Although the immune-mediated therapeutic approaches, based on anti-PD1 or anti-CTLA4, are very promising and under clinical trial experimentation, they could conceal not yet fully emerged pitfalls such as the development of autoimmune diseases. Therefore, alternative therapeutic approaches are still under investigation, such as the immunogenic cell death (ICD) process. Here we show that the lack of calreticulin translocation onto mouse melanoma cell membrane prevents the stimulation of an effective ICD response in vivo.
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http://dx.doi.org/10.1038/s41435-018-0047-7DOI Listing
July 2019

PKR and GCN2 stress kinases promote an ER stress-independent eIF2α phosphorylation responsible for calreticulin exposure in melanoma cells.

Oncoimmunology 2018;7(8):e1466765. Epub 2018 May 31.

Department of Epidemiology, National Institute for Infectious Diseases 'L. Spallanzani', Rome, Italy.

The immunogenic cell death (ICD) process represents a novel therapeutic approach to treat tumours, in which cytotoxic compounds promote both cancer cell death and the emission of damage-associated molecular patterns (DAMPs) from dying cells, to activate the immune system against the malignancy. Therefore, we explored the possibility to stimulate the key molecular players with a pivotal role in the execution of the ICD program in melanoma cells. To this aim, we used the pro-ICD agents mitoxantrone and doxorubicin and found that both agents could induce cell death and stimulate the release/exposure of the strictly required DAMPs in melanoma cells: i) calreticulin (CRT) exposure on the cell membrane; ii) ATP secretion; iii) type I IFNs gene up-regulation and iv) HMGB1 secretion, highlighting no interference by oncogenic BRAF. Importantly, although the ER stress-related PERK activation has been linked to CRT externalization, through the phosphorylation of eIF2α, we found that this stress pathway together with PERK were not involved in melanoma cells. Notably, we identified PKR and GCN2 as key mediators of eIF2α phosphorylation, facilitating the translocation of CTR on melanoma cells surface, under pro-ICD drugs stimulation. Therefore, our data indicate that pro-ICD drugs are able to stimulate the production/release of DAMPs in melanoma cells at least in vitro, indicating in this approach a potential new valuable therapeutic strategy to treat human skin melanoma malignancy.
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http://dx.doi.org/10.1080/2162402X.2018.1466765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136861PMC
May 2018

A functional assay for the clinical annotation of genetic variants of uncertain significance in Diamond-Blackfan anemia.

Hum Mutat 2018 08 28;39(8):1102-1111. Epub 2018 May 28.

Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy.

Diamond-Blackfan anemia (DBA) is a rare genetic hypoplasia of erythroid progenitors characterized by mild to severe anemia and associated with congenital malformations. Clinical manifestations in DBA patients are quite variable and genetic testing has become a critical factor in establishing a diagnosis of DBA. The majority of DBA cases are due to heterozygous loss-of-function mutations in ribosomal protein (RP) genes. Causative mutations are fairly straightforward to identify in the case of large deletions and frameshift and nonsense mutations found early in a protein coding sequence, but diagnosis becomes more challenging in the case of missense mutations and small in-frame indels. Our group recently characterized the phenotype of lymphoblastoid cell lines established from DBA patients with pathogenic lesions in RPS19 and observed that defective pre-rRNA processing, a hallmark of the disease, was rescued by lentiviral vectors expressing wild-type RPS19. Here, we use this complementation assay to determine whether RPS19 variants of unknown significance are capable of rescuing pre-rRNA processing defects in these lymphoblastoid cells as a means of assessing the effects of these sequence changes on the function of the RPS19 protein. This approach will be useful in differentiating pathogenic mutations from benign polymorphisms in identifying causative genes in DBA patients.
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http://dx.doi.org/10.1002/humu.23551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055729PMC
August 2018

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

PLoS One 2018 7;13(2):e0183229. Epub 2018 Feb 7.

RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802440PMC
March 2018

Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

Sci Rep 2017 09 20;7(1):12010. Epub 2017 Sep 20.

Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy.

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.
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http://dx.doi.org/10.1038/s41598-017-12307-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607337PMC
September 2017

Isoforms of the Erythropoietin receptor in dopaminergic neurons of the Substantia Nigra.

J Neurochem 2016 11 30;139(4):596-609. Epub 2016 Sep 30.

Area of Neuroscience, SISSA, Trieste, Italy.

Erythropoietin receptor (EpoR) regulates erythrocytes differentiation in blood. In the brain, EpoR has been shown to protect several neuronal cell types from cell death, including the A9 dopaminergic neurons (DA) of the Substantia Nigra (SN). These cells form the nigrostriatal pathway and are devoted to the control of postural reflexes and voluntary movements. Selective degeneration of A9 DA neurons leads to Parkinson's disease. By the use of nanoCAGE, a technology that allows the identification of Transcription Start Sites (TSSs) at a genome-wide level, we have described the promoter-level expression atlas of mouse A9 DA neurons purified with Laser Capture Microdissection (LCM). Here, we identify mRNA variants of the Erythropoietin Receptor (DA-EpoR) transcribed from alternative TSSs. Experimental validation and full-length cDNA cloning is integrated with gene expression analysis in the FANTOM5 database. In DA neurons, the EpoR gene encodes for a N-terminal truncated receptor. Based on STAT5 phosphorylation assays, we show that the new variant of N-terminally truncated EpoR acts as decoy when co-expressed with the full-length form. A similar isoform is also found in human. This work highlights new complexities in the regulation of Erythropoietin (EPO) signaling in the brain.
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http://dx.doi.org/10.1111/jnc.13757DOI Listing
November 2016

Effects of Pin1 Loss in Hdh(Q111) Knock-in Mice.

Front Cell Neurosci 2016 2;10:110. Epub 2016 May 2.

Department of Health Sciences, University of Piemonte Orientale Novara, Italy.

Huntington's disease (HD) is a fatal, dominantly inherited, neurodegenerative disorder due to a pathological expansion of the CAG repeat in the coding region of the HTT gene. In the quest for understanding the molecular basis of neurodegeneration, we have previously demonstrated that the prolyl isomerase Pin1 plays a crucial role in mediating p53-dependent apoptosis triggered by mutant huntingtin (mHtt) in vitro. To assess the effects of the lack of Pin1 in vivo, we have bred Pin1 knock-out mice with Hdh(Q111) knock-in mice, a genetically precise model of HD. We show that Pin1 genetic ablation modifies a portion of Hdh(Q111) phenotypes in a time-dependent fashion. As an early event, Pin1 activity reduces the DNA damage response (DDR). In midlife mice, by taking advantage of next-generation sequencing technology, we show that Pin1 activity modulates a portion of the alterations triggered by mHtt, extending the role of Pin1 to two additional Hdh(Q111) phenotypes: the unbalance in the "synthesis/concentration of hormones", as well as the alteration of "Wnt/β-catenin signaling". In aging animals, Pin1 significantly increases the number of mHtt-positive nuclear inclusions while it reduces gliosis. In summary, this work provides further support for a role of Pin1 in HD pathogenesis.
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http://dx.doi.org/10.3389/fncel.2016.00110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852193PMC
May 2016

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

Acta Crystallogr D Biol Crystallogr 2015 Nov 31;71(Pt 11):2227-35. Epub 2015 Oct 31.

Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.
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http://dx.doi.org/10.1107/S1399004715015680DOI Listing
November 2015

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome).

RNA Biol 2015 ;12(12):1289-300

a Department of Health Sciences and Interdisciplinary Research Center on Autoimmune Diseases (IRCAD) ; Università del Piemonte Orientale ; Novara , Italy.

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.
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http://dx.doi.org/10.1080/15476286.2015.1107702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829324PMC
October 2016

Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins.

Gene 2015 Sep 2;569(2):287-93. Epub 2015 Jun 2.

Department of Health Science and Interdisciplinary Research Center on Autoimmune Diseases, Università del Piemonte Orientale, Novara, Italy. Electronic address:

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.
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http://dx.doi.org/10.1016/j.gene.2015.05.070DOI Listing
September 2015

SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells.

Front Cell Neurosci 2015 13;9:174. Epub 2015 May 13.

Scuola Internazionale Superiore di Studi Avanzati, Area of Neuroscience Trieste, Italy.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.
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http://dx.doi.org/10.3389/fncel.2015.00174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429562PMC
June 2015

Expression analysis of the long non-coding RNA antisense to Uchl1 (AS Uchl1) during dopaminergic cells' differentiation in vitro and in neurochemical models of Parkinson's disease.

Front Cell Neurosci 2015 1;9:114. Epub 2015 Apr 1.

Area of Neuroscience, International School for Advanced Studies (SISSA) Trieste, Italy.

Antisense (AS) transcripts are RNA molecules that are transcribed from the opposite strand to sense (S) genes forming S/AS pairs. The most prominent configuration is when a lncRNA is antisense to a protein coding gene. Increasing evidences prove that antisense transcription may control sense gene expression acting at distinct regulatory levels. However, its contribution to brain function and neurodegenerative diseases remains unclear. We have recently identified AS Uchl1 as an antisense to the mouse Ubiquitin carboxy-terminal hydrolase L1 (Uchl1) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5. This is mutated in rare cases of early-onset familial Parkinson's Disease (PD) and loss of UCHL1 activity has been reported in many neurodegenerative diseases. Importantly, manipulation of UchL1 expression has been proposed as tool for therapeutic intervention. AS Uchl1 induces UchL1 expression by increasing its translation. It is the representative member of SINEUPs (SINEB2 sequence to UP-regulate translation), a new functional class of natural antisense lncRNAs that activate translation of their sense genes. Here we take advantage of FANTOM5 dataset to identify the transcription start sites associated to S/AS pair at Uchl1 locus. We show that AS Uchl1 expression is under the regulation of Nurr1, a major transcription factor involved in dopaminergic cells' differentiation and maintenance. Furthermore, AS Uch1 RNA levels are strongly down-regulated in neurochemical models of PD in vitro and in vivo. This work positions AS Uchl1 RNA as a component of Nurr1-dependent gene network and target of cellular stress extending our understanding on the role of antisense transcription in the brain.
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http://dx.doi.org/10.3389/fncel.2015.00114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381646PMC
April 2015

An Air-Well sparging minifermenter system for high-throughput protein production.

Microb Cell Fact 2014 Sep 14;13:132. Epub 2014 Sep 14.

Background: Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems.

Results: In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins.

Conclusions: High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.
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http://dx.doi.org/10.1186/s12934-014-0132-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172861PMC
September 2014

Analysis of the interactome of ribosomal protein S19 mutants.

Proteomics 2014 Oct 18;14(20):2286-96. Epub 2014 Sep 18.

Fondazione SDN-IRCCS, Napoli, Italy.

Diamond-Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one-fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild-type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild-type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 (http://proteomecentral.proteomexchange.org/dataset/PXD000640).
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http://dx.doi.org/10.1002/pmic.201300513DOI Listing
October 2014
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