Paolo Tortora | Università degli Studi di Milano-Bicocca (original) (raw)
Papers by Paolo Tortora
Scientific Reports, Mar 6, 2018
The original version of this Article also contained an error in the legend of Figure 5. "Determin... more The original version of this Article also contained an error in the legend of Figure 5. "Determination of ATP levels at different growth times at 30 °C or 37 °C in strains expressing ATX3 variants. Cultures were grown at 30 °C or 37 °C. At the indicated times samples were withdrawn and ATP assayed using the ENLITEN ® ATP Assay System Bioluminescence Detection Kit. Data are expressed as nanomol ATP/OD 600 of cells. Each experiments was performed in technical triplicate. Error bars represent standard deviations and are derived from at least three independent replicates. *p value < 0.01. " now reads: "Determination of ATP levels at different growth times at 30 °C or 37 °C in strains expressing ATX3 variants. Cultures were grown at 30 °C (A) or 37 °C (B). At the indicated times samples were withdrawn and ATP assayed using the ENLITEN ® ATP Assay System Bioluminescence Detection Kit. Data are expressed as nanomol ATP/OD 600 of cells. Each experiments was performed in technical triplicate. Error bars represent standard deviations and are derived from at least three independent replicates. *p value < 0.01. " These errors have now been corrected in the PDF and HTML versions of the Article.
Scientific Reports, Oct 17, 2017
The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neur... more The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation. Ataxin-3 (ATX3) is one among several proteins containing stretches of consecutive glutamines that are responsible for different, albeit related, neurodegenerative diseases in humans, when their size exceeds a critical threshold 1-3. ATX3 triggers the Machado-Joseph disease, an autosomal dominantly inherited neurodegenerative disorder, also referred to as spinocerebellar ataxia type-3. Protein variants carrying polyglutamine (polyQ) stretches whose length exceeds a critical threshold of about 50 consecutive residues, lead to protein misfolding and aggregation into large intracellular inclusions, cytotoxicity and finally dysfunction and demise of specific neurons 4,5. ATX3 consists of a structured globular N-terminal domain, the Josephin domain (JD), followed by a disordered C-terminal tail containing the polyQ stretch, close to the C-terminus 4,6,7. Different physiological roles have been proposed for this protein. Besides a possible role as a modulator of transcription 8-10 , plenty of evidence supports its role as a cysteine protease capable of cleaving isopeptide bonds between ubiquitin (Ub) monomers 11-13. In fact, the JD has the catalytic triad found in these proteases, the residue Cys14 being the one directly involved in catalysis, and displays ubiquitin hydrolase activity even in isolation 11. Furthermore, the C-terminal, disordered domain has two or three ubiquitin-interacting motifs (UIMs) depending on the splice variant 14. However,
Pharmaceutics
Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for... more Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.
International Journal of Molecular Sciences
The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault... more The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We ...
Assessing the toxic effect in living organisms remains a major issue for the development of safe ... more Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer–obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively–we showed that the ideal surface ch...
International Journal of Molecular Sciences
Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, wh... more Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 a...
Advances in High Pressure Bioscience and Biotechnology II, 2003
Protein misfolding and formation of amyloids are the major event in the development of neurodegen... more Protein misfolding and formation of amyloids are the major event in the development of neurodegenerative diseases but the mechanism of this biological phenomenon remains to be elucidated. Here, we report the high pressure denaturation of the spinocerebellar ataxia type 3 protein Ataxin-3 from man and mouse using fluorescence spectroscopy. The two proteins carry 26 and 6 consecutive glutamines, respectively. The tryptophan fluorescence measurements indicated that at pH 7.5 and 25°C the pressure denaturation is reversible but with a large hysteresis in the renaturation profile for both proteins. The reversibility of the pressure-induced unfolding was confirmed by the study of 8-anilinonaphtalene-1-sulfonate (ANS) binding to both proteins. Interestingly, thioflavin-T binding is observed at high pressure, revealing a conversion of the native Ataxin-3 into a potential amyloidogenic state for both proteins. These data provide strong evidence that studying protein folding by hydrostatic pressure may contribute to a better understanding of the mechanism of protein misfolding leading to polyglutamine neurodegenerative diseases.
The International Journal of Biochemistry & Cell Biology, 2007
Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing ... more Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.
PLoS ONE, 2013
Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a mai... more Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity.
Journal of Biological Chemistry, 2003
Journal of Biological Chemistry, 2003
Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to ho... more Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl ␣-NeuAc, ␣233 sialyllactose, glycoproteins (fetuin, ␣-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and ␣233 paragloboside, and vesicular GM3. ␣236 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V max varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (␣ 1-acid glycoprotein), and K m in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V max increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V max values were 25 and 72 IU/mg protein, and K m was 10 and 15 ؋ 10 ؊9 M, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on ␣ 1-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.
Human Molecular Genetics, 2000
Human Gene Therapy, 2001
Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a fir... more Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.
Gene, 1995
This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa r... more This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus I-Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GALl promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3. S. cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the E. coliand Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys 4 and Lys 6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization.
Scientific Reports, Mar 6, 2018
The original version of this Article also contained an error in the legend of Figure 5. "Determin... more The original version of this Article also contained an error in the legend of Figure 5. "Determination of ATP levels at different growth times at 30 °C or 37 °C in strains expressing ATX3 variants. Cultures were grown at 30 °C or 37 °C. At the indicated times samples were withdrawn and ATP assayed using the ENLITEN ® ATP Assay System Bioluminescence Detection Kit. Data are expressed as nanomol ATP/OD 600 of cells. Each experiments was performed in technical triplicate. Error bars represent standard deviations and are derived from at least three independent replicates. *p value < 0.01. " now reads: "Determination of ATP levels at different growth times at 30 °C or 37 °C in strains expressing ATX3 variants. Cultures were grown at 30 °C (A) or 37 °C (B). At the indicated times samples were withdrawn and ATP assayed using the ENLITEN ® ATP Assay System Bioluminescence Detection Kit. Data are expressed as nanomol ATP/OD 600 of cells. Each experiments was performed in technical triplicate. Error bars represent standard deviations and are derived from at least three independent replicates. *p value < 0.01. " These errors have now been corrected in the PDF and HTML versions of the Article.
Scientific Reports, Oct 17, 2017
The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neur... more The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation. Ataxin-3 (ATX3) is one among several proteins containing stretches of consecutive glutamines that are responsible for different, albeit related, neurodegenerative diseases in humans, when their size exceeds a critical threshold 1-3. ATX3 triggers the Machado-Joseph disease, an autosomal dominantly inherited neurodegenerative disorder, also referred to as spinocerebellar ataxia type-3. Protein variants carrying polyglutamine (polyQ) stretches whose length exceeds a critical threshold of about 50 consecutive residues, lead to protein misfolding and aggregation into large intracellular inclusions, cytotoxicity and finally dysfunction and demise of specific neurons 4,5. ATX3 consists of a structured globular N-terminal domain, the Josephin domain (JD), followed by a disordered C-terminal tail containing the polyQ stretch, close to the C-terminus 4,6,7. Different physiological roles have been proposed for this protein. Besides a possible role as a modulator of transcription 8-10 , plenty of evidence supports its role as a cysteine protease capable of cleaving isopeptide bonds between ubiquitin (Ub) monomers 11-13. In fact, the JD has the catalytic triad found in these proteases, the residue Cys14 being the one directly involved in catalysis, and displays ubiquitin hydrolase activity even in isolation 11. Furthermore, the C-terminal, disordered domain has two or three ubiquitin-interacting motifs (UIMs) depending on the splice variant 14. However,
Pharmaceutics
Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for... more Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.
International Journal of Molecular Sciences
The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault... more The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We ...
Assessing the toxic effect in living organisms remains a major issue for the development of safe ... more Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer–obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively–we showed that the ideal surface ch...
International Journal of Molecular Sciences
Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, wh... more Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 a...
Advances in High Pressure Bioscience and Biotechnology II, 2003
Protein misfolding and formation of amyloids are the major event in the development of neurodegen... more Protein misfolding and formation of amyloids are the major event in the development of neurodegenerative diseases but the mechanism of this biological phenomenon remains to be elucidated. Here, we report the high pressure denaturation of the spinocerebellar ataxia type 3 protein Ataxin-3 from man and mouse using fluorescence spectroscopy. The two proteins carry 26 and 6 consecutive glutamines, respectively. The tryptophan fluorescence measurements indicated that at pH 7.5 and 25°C the pressure denaturation is reversible but with a large hysteresis in the renaturation profile for both proteins. The reversibility of the pressure-induced unfolding was confirmed by the study of 8-anilinonaphtalene-1-sulfonate (ANS) binding to both proteins. Interestingly, thioflavin-T binding is observed at high pressure, revealing a conversion of the native Ataxin-3 into a potential amyloidogenic state for both proteins. These data provide strong evidence that studying protein folding by hydrostatic pressure may contribute to a better understanding of the mechanism of protein misfolding leading to polyglutamine neurodegenerative diseases.
The International Journal of Biochemistry & Cell Biology, 2007
Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing ... more Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.
PLoS ONE, 2013
Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a mai... more Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity.
Journal of Biological Chemistry, 2003
Journal of Biological Chemistry, 2003
Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to ho... more Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl ␣-NeuAc, ␣233 sialyllactose, glycoproteins (fetuin, ␣-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and ␣233 paragloboside, and vesicular GM3. ␣236 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V max varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (␣ 1-acid glycoprotein), and K m in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V max increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V max values were 25 and 72 IU/mg protein, and K m was 10 and 15 ؋ 10 ؊9 M, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on ␣ 1-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.
Human Molecular Genetics, 2000
Human Gene Therapy, 2001
Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a fir... more Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.
Gene, 1995
This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa r... more This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus I-Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GALl promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3. S. cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the E. coliand Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys 4 and Lys 6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization.