Publications by authors named "Claudia Scotti"

27 Publications

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In Silico Identification of SOX1 Post-Translational Modifications Highlights a Shared Protein Motif.

Cells 2020 11 13;9(11). Epub 2020 Nov 13.

School of Medicine, The University of Nottingham, Nottingham NG7 2RD, UK.

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer.
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http://dx.doi.org/10.3390/cells9112471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696889PMC
November 2020

High resolution structure of human apolipoprotein (a) kringle IV type 2: beyond the lysine binding site.

J Lipid Res 2020 Dec 9;61(12):1687-1696. Epub 2020 Sep 9.

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.
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http://dx.doi.org/10.1194/jlr.RA120001023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707183PMC
December 2020

HPV Infection Affects Human Sperm Functionality by Inhibition of Aquaporin-8.

Cells 2020 05 17;9(5). Epub 2020 May 17.

Department of Molecular Medicine, Human Physiology Unit, University of Pavia, I-27100 Pavia, Italy.

Human sperm cells express different aquaporins (AQPs), AQP3, 7, 8, 11, which are localized both in the plasma membrane and in intracellular structures. Besides cell volume regulation and end stage of cytoplasm removal during sperm maturation, the role of AQPs extends also to reactive oxygen species (ROS) elimination. Moreover, oxidative stress has been shown to inhibit AQP-mediated HO permeability. A decrease in AQPs functionality is related to a decrease in sperm cells number and motility. Here we investigate the possible effect of human Papillomavirus (HPV) on both expression and function of AQPs in human sperm cells of patients undergoing infertility couple evaluation. Stopped-flow light-scattering experiments demonstrated that HPV infection heavily reduced water permeability of sperm cells in normospermic samples. Confocal immunofluorescence experiments showed a colocalization of HPV L1 protein with AQP8 (Pearson's correlation coefficient of 0.61), confirmed by co-immunoprecipitation experiments. No interaction of HPV with AQP3 and AQP7 was observed. A 3D model simulation of L1 protein and AQP8 interaction was also performed. Present findings may suggest that HPV infection directly inhibits AQP8 functionality and probably makes sperm cells more sensitive to oxidative stress.
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http://dx.doi.org/10.3390/cells9051241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291193PMC
May 2020

Enzymes in Metabolic Anticancer Therapy.

Adv Exp Med Biol 2019 ;1148:173-199

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy.

Cancer treatment has greatly improved over the last 50 years, but it remains challenging in several cases. Useful therapeutic targets are normally unique peculiarities of cancer cells that distinguish them from normal cells and that can be tackled with appropriate drugs. It is now known that cell metabolism is rewired during tumorigenesis and metastasis as a consequence of oncogene activation and oncosuppressors inactivation, leading to a new cellular homeostasis typically directed towards anabolism. Because of these modifications, cells can become strongly or absolutely dependent on specific substrates, like sugars, lipids or amino acids. Cancer addictions are a relevant target for therapy, as removal of an essential substrate can lead to their selective cell-cycle arrest or even to cell death, leaving normal cells untouched. Enzymes can act as powerful agents in this respect, as demonstrated by asparaginase, which has been included in the treatment of Acute Lymphoblastic Leukemia for half a century. In this review, a short outline of cancer addictions will be provided, focusing on the main cancer amino acid dependencies described so far. Therapeutic enzymes which have been already experimented at the clinical level will be discussed, along with novel potential candidates that we propose as new promising molecules. The intrinsic limitations of their present molecular forms, along with molecular engineering solutions to explore, will also be presented.
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http://dx.doi.org/10.1007/978-981-13-7709-9_9DOI Listing
September 2019

HAP1 loss in l-asparaginase resistance.

Blood 2019 05;133(20):2116-2118

University of Pavia.

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http://dx.doi.org/10.1182/blood-2019-03-900993DOI Listing
May 2019

L-asparaginase: A Novel Bacterial Antigen that May Contribute to Infection Detection.

Ann Clin Lab Sci 2018 Sep;48(5):654-658

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy

is responsible for gastric inflammation and for an increased risk of cancer development in humans. Several bacterial antigens contribute to stimulate the immune system, but their relative role has not yet been defined. (strain CCUG) type II L-asparaginase (L-ASNase) induces an immune response in mice. To verify if an immune response could also be detected in humans, sera positive (n=11) or negative (n=11), respectively, to according to a commercial test were assayed for their reactivity towards purified L-ASNase. Among positive samples, 8/11 (72%) were positive to L-ASNase. We conclude that L-ASNase is immunogenic in humans and contributes to the generation of the antibody response induced by the bacterium.
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September 2018

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2.

Int J Biochem Cell Biol 2018 04 5;97:52-61. Epub 2018 Feb 5.

Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV 3, 16132, Genoa, Italy. Electronic address:

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a K of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.
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http://dx.doi.org/10.1016/j.biocel.2018.02.003DOI Listing
April 2018

A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro.

Sci Rep 2017 11 3;7(1):14479. Epub 2017 Nov 3.

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy.

L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.
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http://dx.doi.org/10.1038/s41598-017-15075-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670125PMC
November 2017

Data on enhanced expression and purification of camelid single domain antibodies from classical inclusion bodies.

Data Brief 2017 Jun 31;12:132-137. Epub 2017 Mar 31.

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Via Ferrata, 9, 27100 Pavia, Italy.

Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).
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http://dx.doi.org/10.1016/j.dib.2017.03.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384857PMC
June 2017

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

Protein Expr Purif 2017 Aug 16;136:39-44. Epub 2017 Feb 16.

Department of Molecular Medicine, Unit of Immunology and General Pathology, Via Ferrata, 9, University of Pavia, 27100, Pavia, Italy.

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes.
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http://dx.doi.org/10.1016/j.pep.2017.02.007DOI Listing
August 2017

Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines.

Leuk Res 2015 Jul 22;39(7):757-62. Epub 2015 Apr 22.

Center for Endocrinology, Diabetes & Metabolism, Children's Hospital Los Angeles, Los Angeles, CA, United States; Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States; Department of Physiology & Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States. Electronic address:

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.
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http://dx.doi.org/10.1016/j.leukres.2015.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458142PMC
July 2015

Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase.

Biomolecules 2015 Mar 27;5(2):306-17. Epub 2015 Mar 27.

Unit of Immunology and General Pathology, Department of Molecular Medicine, University of Pavia, Via Ferrata 9, Pavia 27100, Italy.

Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.
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http://dx.doi.org/10.3390/biom5020306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496674PMC
March 2015

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

PLoS One 2015 9;10(2):e0117025. Epub 2015 Feb 9.

Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy.

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117025PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321988PMC
December 2015

Genotype-phenotype correlation in Pompe disease, a step forward.

Orphanet J Rare Dis 2014 Aug 8;9:102. Epub 2014 Aug 8.

Department of Molecular Medicine, University of Pavia, Pavia, Italy.

Background: Pompe's disease is a progressive myopathy caused by mutations in the lysosomal enzyme acid alphaglucosidase gene (GAA). A wide clinical variability occurs also in patients sharing the same GAA mutations, even within the same family.

Methods: For a large series of GSDII patients we collected some clinical data as age of onset of the disease, presence or absence of muscular pain, Walton score, 6-Minute Walking Test, Vital Capacity, and Creatine Kinase. DNA was extracted and tested for GAA mutations and some genetic polymorphisms able to influence muscle properties (ACE, ACTN3, AGT and PPARα genes).We compared the polymorphisms analyzed in groups of patients with Pompe disease clustered for their homogeneous genotype.

Results: We have been able to identify four subgroups of patients completely homogeneous for their genotype, and two groups homogeneous as far as the second mutation is defined "very severe" or "potentially less severe". When disease free life was studied we observed a high significant difference between groups. The DD genotype in the ACE gene and the XX genotype in the ACTN3 gene were significantly associated to an earlier age of onset of the disease. The ACE DD genotype was also associated to the presence of muscle pain.

Conclusions: We demonstrate that ACE and ACTN3 polymorphisms are genetic factors able to modulate the clinical phenotype of patients affected with Pompe disease.
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http://dx.doi.org/10.1186/s13023-014-0102-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249737PMC
August 2014

Activin Receptor-like kinase 1: a novel anti-angiogenesis target from TGF-β family.

Mini Rev Med Chem 2013 Aug;13(10):1398-406

Department of Molecular Medicine - Unit of Immunology and General Pathology University of Pavia- Via Ferrata, 9, 27100-Pavia, Italy.

Anti-angiogenic therapy represents a very promising approach in cancer treatment, as most tumors needs to be supplied by a functional vascular network in order to grow beyond the local boundaries and metastatize. The accessibility of vessels to drug delivery and the broad spectrum of cancers treatable with the same compound have arisen interest in research of suitable molecules, with several, especially targeting the VEGF pathway, entered in clinical trials and approved by the Food and Drug Administration. Despite good results, the major hurdle resides in the limited duration of an effective clinical response before tumors start to grow again. Thus, researchers are looking for different alternative targets for a combined and parallel multi-targeting of angiogenic signaling circuits. Activin Receptor-like kinase 1 (ALK1) is a TGF-β type I receptor with high affinity for the BMP9 member of Bone Morphogenic Proteins superfamily: it is expressed mainly, even if not exclusively, on endothelial cells and seems to be involved in the regulatory phase of angiogenesis. Despite a non-completely elucidated mechanism, the targeting of this pathway, both by a soluble ALK1-Fc receptor developed by Acceleron Pharma and by a fully human monoclonal antibody developed by Pfizer, has achieved encouraging results. After having briefly summarized the state of the art of anti-angiogenic therapy, we will first review existing evidence about the molecular mechanisms of ALK1 signaling and we will then analyse in detail the pre-clinical and clinical data available about these two drugs.
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http://dx.doi.org/10.2174/13895575113139990065DOI Listing
August 2013

Bioinformatic analysis of pathogenic missense mutations of activin receptor like kinase 1 ectodomain.

PLoS One 2011 18;6(10):e26431. Epub 2011 Oct 18.

Department of Experimental Medicine, Section of General Pathology, University of Pavia, Pavia, Italy.

Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1(EC)) has been elusive so far. We here describe the building of a homology model for ALK1(EC), followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1(EC) potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1(EC) allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1(EC) and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026431PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196573PMC
February 2012

Targeted drug delivery using immunoconjugates: principles and applications.

J Immunother 2011 Nov-Dec;34(9):611-28

Department of Experimental Medicine, Section of General Pathology, University of Pavia, Via Ferrata, Italy.

Antibody-drug conjugates (also known as "immunoconjugates") have only recently entered the arsenal of anticancer drugs, but the number of undergoing clinical trials including them is ever increasing and most therapeutic antibodies are now patented including their potential immunoconjugate derivatives. They typically consist of three components: antibody, linker, and cytotoxin. An antibody or antibody fragment targeted to a tumor-associated antigen acts as a carrier for drug delivery and can be conjugated by cleavable or uncleavable linkers to a variety of effector molecules, either a drug, toxin, radioisotope, enzyme (the latter also used in Antibody-Directed Enzyme Prodrug Therapy), or to drug-containing liposomes or nanoparticles. In this review, we propose a general outline of the field, starting from the diagnostic and clinical applications of this class of molecules. Special attention will be devoted to the principles and issues in molecular design (choice of tumor-associated antigen, critical milestones in antibody development, available alternatives for linkers and effector molecule, and strategies for fusion proteins building) to the importance of antibody affinity modulation to optimize therapeutic effect and the potential of emerging alternative scaffolds. Most of the power of these molecules is to reach high concentrations in the tumor, relatively unaffecting normal cells, although one drawback lies in their short half-life. In this respect, modifications of immunoconjugates, which have shown to strongly influence pharmacokinetics, like glycosylation and PEGylation, will be discussed. Undergoing clinical trials and active patents will be analyzed and problems present in clinical use will be reported.
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http://dx.doi.org/10.1097/CJI.0b013e318234ecf5DOI Listing
February 2012

Expanding targets for a metabolic therapy of cancer: L-asparaginase.

Recent Pat Anticancer Drug Discov 2012 Jan;7(1):4-13

Department of Experimental Medicine, Section of General Pathology, University of Pavia, Via Ferrata, 1, 27100 Pavia, Italy.

The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.
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http://dx.doi.org/10.2174/157489212798358001DOI Listing
January 2012

Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

PLoS One 2010 Nov 9;5(11):e13892. Epub 2010 Nov 9.

Department of Experimental Medicine, Section of General Pathology, University of Pavia, Pavia, Italy.

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013892PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976697PMC
November 2010

Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent.

Biochem Biophys Res Commun 2008 Dec 5;377(4):1222-6. Epub 2008 Nov 5.

Department of Experimental Medicine, Section of General Pathology, University of Pavia, Piazza Botta, 10, 27100 Pavia, Italy.

Bacterial L-asparaginases are amidohydrolases that catalyse the conversion of L-asparagine to L-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L-asparagine over L-glutamine and, in contrast to most L-asparaginases, it exhibited a sigmoidal behaviour towards L-glutamine. The enzyme preserved full activity after 2 h incubation at 45 degrees C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.
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http://dx.doi.org/10.1016/j.bbrc.2008.10.118DOI Listing
December 2008

Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Jan 16;63(Pt 1):18-20. Epub 2006 Dec 16.

DiSCAFF&DFB Center, Università del Piemonte Orientale A. Avogadro, Via Giovanni Bovio 6, 28100, Novara, Italy.

The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell-cell and cell-matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.
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http://dx.doi.org/10.1107/S1744309106051682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330100PMC
January 2007

Structural basis of affinity maturation of the TEPC15/Vkappa45.1 anti-2-phenyl-5-oxazolone antibodies.

J Mol Biol 2006 Jun 2;359(5):1161-9. Epub 2006 May 2.

Dipartimento di Medicina Sperimentale, Universita' di Pavia, Italy.

Affinity maturation is a process that leads to the emergence of more efficient antibodies following initial antigen encounter and represents a key strategy of the adaptive immunity of vertebrate organisms. Earlier and detailed sequence studies of the antibody response to a model antigen, the hapten 2-phenyl-5-oxazolone (phOx), define three different classes of antibodies. Class I antibodies use the V(H)Ox1/V(kappa)Ox1 gene pair and dominate the early stages of the anti-phOx response, class II antibodies use the V(kappa)Ox1 gene but a different V(H) segment and are common in the intermediate stages, and class III antibodies use the TEPC15/V(kappa)45.1 genes and play the greatest role in the late stages. Only the crystal structure of one anti-phOx antibody, the class II NQ10/12.5 Fab fragment, has been described. Here we report the crystal structures of the scFv form of the low and high affinity anti-phOx class III antibodies NQ10/1.12 and NQ16/113.8 complexed with the hapten. The two antibodies differ by nine amino acid substitutions, all located in the V(H) domain. Analysis of the two structures shows that affinity maturation results from an increase in surface complementarity, as a consequence of a finely tuned and highly concerted process chaperoned by the somatic mutations, and implies a more efficient hapten-induced fit in the mature antibody. The data also demonstrate that class III antibodies respond in a completely different way to the architectural problem of binding phOx compared to the class II antibody NQ10/12.5.
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http://dx.doi.org/10.1016/j.jmb.2006.04.036DOI Listing
June 2006

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria.

Mitochondrion 2003 Apr;2(5):361-73

Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale, Università di Pavia, Piazza Botta 10, 27100 Pavia, Italy.

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.
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http://dx.doi.org/10.1016/S1567-7249(03)00004-7DOI Listing
April 2003

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression.

Exp Cell Res 2002 Nov;281(1):128-39

Dipartimento di Medicina Sperimentale, sez. Patologia generale C. Golgi, Universita' di, Pavia, Italy.

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.
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http://dx.doi.org/10.1006/excr.2002.5629DOI Listing
November 2002