Daria Maria Monti

Effects of the Known Pathogenic Mutations on the Aggregation Pathway of the Amyloidogenic Peptide of Apolipoprotein A-I

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression.

Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells.

Recent years have witnessed an unprecedented growth in the number of applications—such as drug delivery, nutraceuticals and production of improved biocompatible materials—in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44 nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent.

The cytosolic ribonuclease inhibitor contributes to intracellular redox homeostasis

The hypothesis that the cytosolic RNase inhibitor (cRI) has a role in the protection of the cellular redox homeostasis was investigated testing the effects of oxidants and anti‐oxidants on normal, primary endothelial HUVE cells, and malignant HeLa cells, before and after their engineering into cRI‐deprived cells. We found that cRI plays an important, possibly a key, physiological role in the protection of cells from redox stress, as demonstrated by decreased GSH levels as well as increased oxidant‐induced DNA damage in cRI deprived cells.

Human carbonic anhydrase VII protects cells from oxidative damage.

Abstract Human carbonic anhydrase (hCA) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Recently, S-glutathionylation of two cysteine residues from the enzyme was revealed, suggesting a new role as oxygen radical scavenger. We analyzed the effect of native and tetramutated hCA VII (all cysteines mutated into serines) in a eukaryotic system by stressing cells with an oxidant agent. Results clearly show that native hCA VII can protect cells from oxidative damage by preventing the apoptosis cascade and that cysteines play a leading role in this process. Our findings definitively confirm hCA VII protective role toward oxidative insult.

An ascorbic acid-enriched tomato genotype to fight UVA-induced oxidative stress in normal human keratinocytes

UVA radiations contribute up to 95% of the total UV exposure and are known to induce cell damage, leading to apoptosis. Since the benefic effects of ascorbic acid on human health are well known, a new tomato genotype (named DHO4), highly rich in ascorbic acid, has been recently obtained. Here, we compared the effects of ascorbic acid and hydrophilic DHO4 extracts in protecting human keratinocytes exposed to UVA stress. Keratinocytes were pre-incubated with ascorbic acid or with extracts from the ascorbic acid enriched tomato genotype and irradiated with UVA light. Then, ROS production, intracellular GSH and lipid peroxidation levels were quantified. Western blots were carried out to evaluate mitogen-activated protein kinases cascade, activation of caspase-3 and inflammation levels. We demonstrated that ROS, GSH and lipid peroxidation levels were not altered in cell exposed to UVA stress when cells were pre-treated with ascorbic acid or with tomato extracts. In addition, no evidence of apoptosis and inflammation were observed in irradiated pre-treated cells. Altogether, we demonstrated the ability of an ascorbic acid enriched tomato genotype to counteract UVA-oxidative stress on human keratinocytes. This protective effect is due to the high concentration of vitamin C that acts as free radical scavenger. This novel tomato genotype may be used as genetic material in breeding schemes to produce improved varieties with higher antioxidant levels.

Quantitative Trait Loci Pyramiding Can Improve the Nutritional Potential of Tomato (Solanum lycopersicum) Fruits

Solanum lycopersicum represents an important source of antioxidants and other bioactive compounds. Previously two Solanum pennellii introgression lines (IL 7-3 and IL 12-4) were identified as carrying quantitative trait loci (QTL) increasing fruit ascorbic acid and phenolics content. Novel tomato lines were obtained by pyramiding these selected QTLs in the genetic background of the cultivated line M82. Pyramided lines revealed significant increases of total phenolics, phenolic acids, ascorbic acid, and total antioxidant activity compared to parental lines IL 7-3 and IL 12-4 and the cultivated line M82. In addition, tomato extracts obtained from the pyramided lines had no cytotoxic effect on normal human cells while exhibiting a selective cytotoxic effect on aggressive cancer cells. Therefore, the present study demonstrates that it is possible to incorporate favorable wild-species QTLs in the cultivated genetic background to obtain genotypes with higher nutritional value.

Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells

Apolipoprotein A‐I (ApoA‐I) is an extracellular lipid acceptor, whose role in cholesterol efflux and high‐density lipoprotein formation is mediated by ATP‐binding cassette transporter A1 (ABCA1). Nevertheless, some ApoA‐I variants are associated to systemic forms of amyloidosis, characterized by extracellular fibril deposition in peripheral organs. Heart amyloid fibrils were found to be mainly constituted by the 93‐residue N‐terminal fragment of ApoA‐I, named [1–93]ApoA‐I. In this paper, rat cardiomyoblasts were used as target cells to analyse binding, internalization and intracellular fate of the fibrillogenic polypeptide in comparison to full‐length ApoA‐I. We provide evidence that the polypeptide: (i) binds to specific sites on cell membrane (Kd= 5.90 ± 0.70 × 10−7 M), where it partially co‐localizes with ABCA1, as also described for ApoA‐I; (ii) is internalized mostly by chlatrin‐mediated endocytosis and lipid rafts, whereas ApoA‐I is internalized preferentially by chlatrin‐coated pits and macropinocytosis and (iii) is rapidly degraded by proteasome and lysosomes, whereas ApoA‐I partially co‐localizes with recycling endosomes. Vice versa, amyloid fibrils, obtained by in vitro aggregation of [1–93]ApoA‐I, were found to be unable to enter the cells. We propose that internalization and intracellular degradation of [1–93]ApoA‐I may divert the polypeptide from amyloid fibril formation and contribute to the slow progression and late onset that characterize this pathology.

Enzymatically active fibrils generated by the self-assembly of the ApoA-I fibrillogenic domain functionalized with a catalytic moiety

Enzymatically active fibrils were produced by self-assembly of a bifunctional chimeric protein, made up of a fibrillogenic and a catalytic moiety. For this purpose, the fibrillogenic domain of Apolipoprotein A-I (ApoA-I), a 93-residue polypeptide named [1-93]ApoA-I, was functionalized with the enzyme glutathione S-transferase (GST). The fusion protein GST-[1-93]ApoA-I was expressed, isolated to homogeneity and characterized. In the soluble form, GST-[1-93]ApoA-I was found to be fully active as a GST enzyme, and to have high propensity to self-aggregate. Upon incubation for 3 weeks at pH 6.4, insoluble aggregates were generated. Analyzed by AFM, they were found to contain fibrillar structures often organized into large fiber networks. Fibrils were loaded on the membrane of a microfiltration unit and tested for enzymatic activity by filtering the substrate through the fibrillar network. Fibrils were shown to be catalytically active, stable over time and reusable, as no loss of activity was detected when fibrils were repeatedly tested. Our findings suggest that catalytically active fibrils may be of interest for biocatalytic applications in nanobiotechnology.

Protein conformational perturbations in hereditary amyloidosis: Differential impact of single point mutations in ApoAI amyloidogenic variants

Amyloidoses are devastating diseases characterized by accumulation of misfolded proteins which aggregate in fibrils. Specific gene mutations in Apolipoprotein A I (ApoAI) are associated with systemic amyloidoses. Little is known on the effect of mutations on ApoAI structure and amyloid properties. Here we performed a physico-chemical characterization of L75P- and L174S-amyloidogenic ApoAI (AApoAI) variants to shed light on the effects of two single point mutations on protein stability, proteolytic susceptibility and aggregation propensity. Both variants are destabilized in their N-terminal region and generate fibrils with different morphological features. L75P-AApoAI is significantly altered in its conformation and compactness, whereas a more flexible and pronounced aggregation-competent state is associated to L174S-AApoAI. These observations point out how single point mutations in ApoAI gene evocate differences in the physico-chemical and conformational behavior of the corresponding protein variants, with the common feature of diverting ApoAI from its natural role towards a pathogenic pathway.

Insights into the role of reactive sulfhydryl groups of carbonic anhydrase III and VII during oxidative damage

Abstract Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.