Dipartimento di Biotecnologie e Bioscienze

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.

We present the synthesis of trastuzumab-functionalized pegylated iron oxide nanoparticles and provide an FTIR-based approach to gain a direct evidence of the actual conservation of the native structure of conjugated antibody. Their target-selectivity to specific cancer cell receptors has been also assessed.

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291D). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291D strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291D on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular b-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

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.

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an antioligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm 21 in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.

Aggregation of human ataxin-3 (AT3) into amyloid fibrils is responsible for spinocerebellar ataxia type 3. This protein consists of a folded N-terminal domain (Josephin domain, residues 1-182), a central flexible region (residues 183-291), a poly-glutamine sequence of variable length and a short C-terminal flexible region. Very little is known about the influence of the central flexible region on the conformational and aggregation properties of this protein. The present study aimed to investigate the specific role of this portion of the protein (residues 183-291). Accordingly, protein fragments 1-182 (AT3 ⁄ 182) and 1-291 (AT3 ⁄ 291) were produced and compared by thioflavin-T fluorescence, Fourier transform infrared spectroscopy, CD, intrinsic fluorescence and ESI-MS. It is shown that the central flexible region enhances protein aggregation and can populate conformational states with different degrees of compactness. Both monomeric and dimeric partially-folded forms are identified for both protein fragments under denaturing conditions. Partially-folded monomers and dimers accumulate to a larger extent in AT3 ⁄ 291. These species represent good candidates for early intermediates of the aggregation process under the experimental conditions employed in the present study.

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid β-rich fibrils, whose hallmark is the...

Proteins from hyperthermophilic microorganisms are generally capable of withstanding temperatures close to, or even higher than the boiling point. As a rule, these proteins are strongly piezostable as well, although exceptions have been also reported. This observation has a theoretical relevance, as the understanding of the effects of pressure and temperature on protein stability is equally important to develop a comprehensive model for their thermodynamic stability. Nevertheless, the structural features justifying the correlation between heat resistance and pressure resistance are poorly understood. Actually, most reports do not exceed the phenomenological level. Only in the case of the small protein Sso7d from Sulfolobus solfataricus, characterisation of wild-type and some mutants showed that both properties are largely accounted for by a network of aromatic residues found in the hydrophobic core of the molecule. Current knowledge, however, does not allow to establish to what extent this finding may be generalised. In a biotechnological perspective, hyperthermophilic enzymes seem to be more suitable for bioprocesses at high pressure with respect to their mesophilic counterparts. Indeed, thanks to their higher resistance towards pressure and temperature, they may be exploited in a much broader range of working conditions for tuning activity and specificity. Furthermore, they are often activated by increasing pressure, although it cannot be established, to date, to what extent this is a common feature. ß

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100°C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54∆). It is shown that L54∆ has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N S D transition model, with a denaturation temperature of 53°C. Fluorescence titration experiments indicate that L54∆ binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.