Silvia Maria Doglia

Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy

The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O. The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of alpha-helices. We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding. The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity. Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200-900 cm(-1) carbohydrate region. A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein. To confirm that the FTIR absorption in the 1200-900 cm(-1) region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties. Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.

Quantitative study of doxorubicin in living cell nuclei by microspectrofluorometry

Doxorubicin-DNA association has been studied by quantitative microspectrofluorometry. Fluorescence emission spectra from a microvolume of single living cell nuclei treated with doxorubicin have been analyzed in terms of difference in spectral shape and fluorescence yield between free and DNA-bound drug. Contribution of each spectral component to the total signal was calculated by least-squares linear regression. With this method of analysis, total drug concentration has been determined with an error of less than 10%. Moreover, the uptake into the nucleus has been studied in a non destructive way, avoiding use of 14C-labelled drug. Kinetic studies of drug accumulation into the nuclei were conducted on sensitive and resistant cells.

Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures.

The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering. We investigated the self-assembled nanostructures of functionalized peptides featuring four, two or no glycine-spacers between the self-assembly sequence RADA16-I and the functional biological motif PFSSTKT. The effectiveness of their biological functionalization was assessed via in vitro experiments with neural stem cells (NSCs) and their molecular assembly was elucidated via atomic force microscopy, Raman and Fourier Transform Infrared spectroscopy. We demonstrated that glycine-spacers play a crucial role in the scaffold stability and in the exposure of the functional motifs. In particular, a glycine-spacer of four residues leads to a more stable nanostructure and to an improved exposure of the functional motif. Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation. Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

Tetracycline and its analogues protect Caenorhabditis elegans from β amyloid-induced toxicity by targeting oligomers.

The accumulation and deposition of amyloid beta (Aβ) peptide in extracellular dense plaques in the brain is a key phase in Alzheimers disease (AD). Small oligomeric forms of Aβ are responsible for the toxicity and the early cognitive impairment observed in patients before the amyloid plaque deposits appear. It is essential for the development of an efficient cure for AD to identify compounds that interfere with Aβ aggregation, counteracting the molecular mechanisms involved in conversion of the monomeric amyloid protein into oligomeric and fibrillar forms. Tetracyclines have been proposed for AD therapy, although their effects on the aggregation of Aβ protein, particularly their ability to interact in vivo with the Aβ oligomers and/or aggregates, remain to be understood. Using transgenic Caenorhabditis elegans as a simplified invertebrate model of AD, we evaluated the ability of tetracyclines to interfere with the sequence of events leading to Aβ proteotoxicity. The drugs directly interact with the Aβ assemblies in vivo and reduce Aβ oligomer deposition, protecting C. elegans from oxidative stress and the onset of the paralysis phenotype. These effects were specific, dose-related and not linked to any antibiotic activity, suggesting that the drugs might offer an effective therapeutic strategy to target soluble Aβ aggregates.

Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy.

The aggregation of a recombinant lipase as inclusion bodies (IBs) was studied directly within intact Escherichia coli cells by FT‐IR microspectroscopy. Through this approach, it was possible to monitor in real time the different kinetics of IB formation at 37 and 27 °C, in excellent agreement with the results of the SDS–PAGE analysis. Furthermore, insights on the residual native‐like structure of the expressed protein within IB – both isolated and inside cells – were obtained by the secondary structure analysis of the Amide I band in the IB FT‐IR spectra.

BMHP1-Derived Self-Assembling Peptides: Hierarchically Assembled Structures with Self-Healing Propensity and Potential for Tissue Engineering Applications

Self-assembling peptides (SAPs) are rapidly gaining interest as bioinspired scaffolds for cell culture and regenerative medicine applications. Bone Marrow Homing Peptide 1 (BMHP1) functional motif (PFSSTKT) was previously demonstrated to stimulate neural stem cell (NSC) viability and differentiation when linked to SAPs. We here describe a novel ensemble of SAPs, developed from the BMHP1 (BMHP1-SAPs), that spontaneously assemble into tabular fibers, twisted ribbons, tubes and hierarchical self-assembled sheets: organized structures in the nano- and microscale. Thirty-two sequences were designed and evaluated, including biotinylated and unbiotinylated sequences, as well as a hybrid peptide-peptoid sequence. Via X-ray diffraction (XRD), CD, and FTIR experiments we demonstrated that all of the BMHP1-SAPs share similarly organized secondary structures, that is, β-sheets and β-turns, despite their heterogeneous nanostructure morphology, scaffold stiffness, and effect over NSC differentiation and survival. Notably, we demonstrated the self-healing propensity of most of the tested BMHP1-SAPs, enlarging the set of potential applications of these novel SAPs. In in vitro cell culture experiments, we showed that some of these 10-mer peptides foster adhesion, differentiation, and proliferation of human NSCs. RGD-functionalized and hybrid peptide-peptoid self-assembling sequences also opened the door to BMHP1-SAP functionalization with further bioactive motifs, essential to tailor new scaffolds for specific applications. In in vivo experiments we verified a negligible reaction of the host nervous tissue to the injected and assembled BMHP1-SAP. This work will pave the way to the development of novel SAP sequences that may be useful for material science and regenerative medicine applications.

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co‐expression of chaperones.

Regulation of cellular glutathione modulates nuclear accumulation of daunorubicin in human MCF7 cells overexpressing multidrug resistance associated protein.

Multidrug resistance (MDR) is frequently associated with the overexpression of P-glycoprotein (Pgp) and/or multidrug resistance associated protein (MRP1), both members of the ABC superfamily of transporters. Pgp and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from tumour cells. Glutathione (GSH) has been considered to play an important role in the MRP1-mediated MDR. In our study, we examined the effects of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, on the nuclear accumulation of daunorubicin (DNR), in etoposide (VP16) and doxorubicin (ADR) resistant MCF7 cell lines, overexpressing respectively MRP1 (MCF7/VP) and Pgp (MCF7/ADR). The study of DNR transport was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of the nuclear accumulation of anthracyclines in single living tumour cells. Treatment of MCF7/VP cells with BSO increased the sensitivity of these cells to DNR whilst the cytotoxicity of the drug in MCF7/ADR cells remained unchanged. In MCF7 resistant cells treated with BSO, their GSH level decreased as observed by confocal microscopy. DNR nuclear accumulation in MCF7/VP cells was increased by BSO whereas in MCF7/ADR cells BSO was unable to significantly increase the DNR nuclear accumulation. These data suggest a requirement for GSH in MRP1-mediated resistance whilst the nuclear efflux of GSH conjugates is probably not the primary mechanism of Pgp-mediated MDR. Finally, BSO might be a useful agent in clinical assays for facilitating detection of MRP1 expression.

A Major Role for Side-Chain Polyglutamine Hydrogen Bonding in Irreversible Ataxin-3 Aggregation

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 anti-oligomer 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−1 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.

Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: Chemical characterization and evaluation of the protein adsorption

This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples. The efficiency of these processes was evaluated in terms of the amount of grafted polymer, coverage uniformity and substrates wettability. The process was monitored by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and atomic force microscopy (AFM) analyses. The stability of the obtained thin films was evaluated in water and in Phosphate Buffer Saline (PBS) at 37 degrees C. The adsorption of fibrinogen and green fluorescent protein (GFP)--taken as model proteins--on the differently prepared surfaces was evaluated through a fluorescence approach using laser scanning confocal microscopy with photon counting detection. After plasma treatments of short duration, the protein adsorption decreases by about 60-70% with respect to that of the untreated film, while long plasma exposure resulted in a higher protein adsorption, due to damaging of the grafted polymer.