Publications by authors named "Alessandro Roncador"

11 Publications

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Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aβ1-42-mediated toxicity.

Neurobiol Dis 2020 07 25;140:104849. Epub 2020 Mar 25.

Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, TN, Italy. Electronic address:

Alzheimer's disease (AD) is the most common cause of dementia. At the pre-symptomatic phase of the disease, the processing of the amyloid precursor protein (APP) produces toxic peptides, called amyloid-β 1-42 (Aβ 1-42). The downstream effects of Aβ 1-42 production are not completely uncovered. Here, we report the involvement of transglutaminase 1 (TG1) in in vitro AD models of neuronal toxicity. TG1 was increased at late stages of the disease in the hippocampus of a mouse model of AD and in primary cortical neurons undergoing stress. Silencing of TGM1 gene was sufficient to prevent Aβ-mediated neuronal death. Conversely, its overexpression enhanced cell death. TGM1 upregulation was mediated at the transcriptional level by an activator protein 1 (AP1) binding site that when mutated halted TGM1 promoter activation. These results indicate that TG1 acts downstream of Aβ-toxicity, and that its stress-dependent increase makes it suitable for pharmacological intervention.
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http://dx.doi.org/10.1016/j.nbd.2020.104849DOI Listing
July 2020

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons.

J Cell Sci 2019 09 26;132(18). Epub 2019 Sep 26.

Laboratory of Molecular and Cellular Neurobiology, Department of Cellular, Computational and Integrative Biology-CIBIO, University of Trento, 38123 Trento, Italy

Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.222802DOI Listing
September 2019

Primary cortical neurons on PMCS TiO films towards bio-hybrid memristive device: A morpho-functional study.

Biophys Chem 2017 10 26;229:115-122. Epub 2017 Apr 26.

Istituto di Biofisica, Unità di Trento, Consiglio Nazionale delle Ricerche (IBF-CNR), Via alla Cascata 56/C 18, 38123 Trento, Italy.

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO) prepared by Pulsed Microplasma Cluster Source (PMCS). This technique uses supersonic pulsed beams seeded by clusters of the metal oxide synthesized in a plasma discharge. The final stoichiometry of the TiO thin films is tuned changing the gas mixture, achieving stoichiometric or oxygen overstoichiometric films. All the films showed consistent biocompatibility and a spontaneous absorption of poly-d-lysine (PDL) that favors the adhesion and growth of murine cortical neurons. Moreover, the bioelectrical activity of the neuronal culture grown on the TiO film can be modulated by changing the chemistry of the surface. This work paves the way to develop a bio-hybrid neuromorphic device, where viable nerve cells are grown directly over a titanium dioxide film showing a network of memristors.
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http://dx.doi.org/10.1016/j.bpc.2017.04.010DOI Listing
October 2017

Use of polymer conjugates for the intraperoxisomal delivery of engineered human alanine:glyoxylate aminotransferase as a protein therapy for primary hyperoxaluria type I.

Nanomedicine 2017 04 18;13(3):897-907. Epub 2016 Dec 18.

Neuroscience, Biomedicine and Movement Sciences Department, Section of Biological Chemistry, University of Verona, Verona (VR), Italy. Electronic address:

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficit causes the rare disorder Primary Hyperoxaluria Type I (PH1). We now describe the conjugation of poly(ethylene glycol)-co-poly(L-glutamic acid) (PEG-PGA) block-co-polymer to AGT via the formation of disulfide bonds between the polymer and solvent-exposed cysteine residues of the enzyme. PEG-PGA conjugation did not affect AGT structural/functional properties and allowed the enzyme to be internalized in a cellular model of PH1 and to restore glyoxylate-detoxification. The insertion of the C387S/K390S amino acid substitutions, known to favor interaction with the peroxisomal import machinery, reduced conjugation efficiency, but endowed conjugates with the ability to reach the peroxisomal compartment. These results, along with the finding that conjugates are hemocompatible, stable in plasma, and non-immunogenic, hold promise for the development of polypeptide-based AGT conjugates as a therapeutic option for PH1 patients and represent the base for applications to other diseases related to deficits in peroxisomal proteins.
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http://dx.doi.org/10.1016/j.nano.2016.12.011DOI Listing
April 2017

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

Biochim Biophys Acta 2015 Oct 3;1854(10 Pt A):1280-9. Epub 2015 Jul 3.

Department of Life and Reproduction Sciences, University of Verona, Verona, Italy. Electronic address:

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.
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http://dx.doi.org/10.1016/j.bbapap.2015.07.002DOI Listing
October 2015

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation.

Hum Mol Genet 2014 Nov 2;23(22):5998-6007. Epub 2014 Jul 2.

Department of Life Sciences and Reproduction, University of Verona, Verona, Italy and.

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5'-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.
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http://dx.doi.org/10.1093/hmg/ddu329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204775PMC
November 2014

Allele-specific characterization of alanine: glyoxylate aminotransferase variants associated with primary hyperoxaluria.

PLoS One 2014 9;9(4):e94338. Epub 2014 Apr 9.

Department of Biology, Duke University, Durham, NC, USA; Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, CO, USA.

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094338PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981788PMC
June 2015

Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase.

Biochim Biophys Acta 2013 Dec 17;1832(12):2277-88. Epub 2013 Sep 17.

Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona, Strada Le Grazie 8 37134 Verona, Italy.

Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.
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http://dx.doi.org/10.1016/j.bbadis.2013.09.002DOI Listing
December 2013

S250F variant associated with aromatic amino acid decarboxylase deficiency: molecular defects and intracellular rescue by pyridoxine.

Hum Mol Genet 2013 Apr 15;22(8):1615-24. Epub 2013 Jan 15.

Department of Life Sciences and Reproduction, University of Verona, Verona, Italy.

Dopa or aromatic amino acid decarboxylase (DDC, AADC) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the production of the neurotransmitters dopamine and serotonin. Among the so far identified mutations associated with AADC deficiency, an inherited rare neurometabolic disease, the S250F mutation is the most frequent one. Here, for the first time, the molecular basis of the deficit of the S250F variant was investigated both in vitro and in cellular systems. Ser250 is not essential for the catalytic activity of the enzyme. However, its mutation to Phe causes a ~7-fold reduction of catalytic efficiency and a conformational change in the proximity of the mutated residue that is transmitted to the active site. In cellular extracts of E. coli and mammalian cells, both the specific activity and the protein level of the variant decrease with respect to the wild-type. The results with mammalian cells indicate that the mutation does not affect intracellular mRNA levels, and are consistent with a model where S250F undergoes a degradation process via the proteasome, possibly through an ubiquitination process occurring faster than in the wild-type. Overall, biochemical and cell biology experiments show that loss of function of S250F occurs by two distinct but not exclusive mechanisms affecting activity and folding. Importantly, 4-phenylbutirric acid (4-PBA) or, to a major extent, pyridoxine increase the expression level and, in a dose-dependent manner, the decarboxylase specific activity of mutant-expressing cells. This strongly suggests that 4-PBA and/or pyridoxine administration may be of important value in therapy of patients bearing the S250F mutation.
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http://dx.doi.org/10.1093/hmg/ddt011DOI Listing
April 2013

Rapid profiling of disease alleles using a tunable reporter of protein misfolding.

Genetics 2012 Nov 24;192(3):831-42. Epub 2012 Aug 24.

Department of Biology, Duke University, Durham, North Carolina 27708, USA.

Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.
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http://dx.doi.org/10.1534/genetics.112.143750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522161PMC
November 2012

K644E/M FGFR3 mutants activate Erk1/2 from the endoplasmic reticulum through FRS2 alpha and PLC gamma-independent pathways.

J Mol Biol 2006 Mar 3;357(3):783-92. Epub 2006 Feb 3.

Division of Biochemistry, Department of Morphological and Biomedical Sciences, University of Verona Medical School, 37134 Verona, Italy.

Fibroblast growth factor receptors 3 (FGFR3) with K644M/E substitutions are associated to the severe skeletal dysplasias: severe achondroplasia with developmental delay and achanthosis nigricans(SADDAN) and thanatophoric dysplasia(TDII). The high levels of kinase activity of the FGFR3-mutants cause uncompleted biosynthesis that results in the accumulation of the immature/mannose-rich, phosphorylated receptors in the endoplasmic reticulum (ER) and STATs activation. Here we report that FGFR3 mutants activate Erk1/2 from the ER through an FRS2-independent pathway: instead, a multimeric complex by directly recruiting PLCgamma, Pyk2 and JAK1 is formed. The Erk1/2 activation from the ER however, is PLCgamma-independent, since preventing the PLCgamma/FGFR3 interaction by the Y754F substitution does not inhibit Erks. Furthermore, Erk1/2 activation is abrogated upon treatment with the Src inhibitor PP2, suggesting a role played by a Src family member in the pathway from the ER. Finally we show that the intrinsic kinase activity by mutant receptors is required to allow signaling from the ER. Overall these results highlight how activated FGFR3 exhibits signaling activity in the early phase of its biosynthesis and how segregation in a sub-cellular compartment can affect the FGFR3 multi-faceted capacity to recruit specific substrates.
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http://dx.doi.org/10.1016/j.jmb.2006.01.058DOI Listing
March 2006