Carrotta R

Toxicity of recombinant β-amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus

A distinctive feature of Alzheimers disease is the deposition of amyloid β‐protein (Aβ) in senile or diffuse plaques. The 42 residue β‐peptide (Aβ42) is the predominant form found in plaques. In the present work we report a high‐yield expression and purification method of production of a recombinant Aβ42. The purified recombinant peptide shows characteristics similar to the synthetic human peptide. Different size aggregates, either small oligomers or larger aggregates, were obtained upon dissolving the recombinant Aβ42 peptide under different conditions at pH 7.2 or pH 3, respectively. We report a new toxicity assay on the morphogenic development of the sea urchin Paracentrotus lividus and study the toxicity of the two kinds of aggregates. Despite the difference between the ionic strength of human extracellular fluid (0.154 mol/l) and artificial sea water (0.48 mol/l), toxicity data collected in this system have an intrinsic relevance. The different ionic strength, in fact, could change the kinetics of oligomer formation, but the effect of morphogenic development reported here is related to the final oligomer sizes. Results of the toxicity assay of Aβ42 on sea urchin development also show a dose‐dependent effect. After only 4 h of embryo development, one can note morphological defects in the cell membrane. Retardation of the embryos development, along with cellular disorders visible inside the blastocoele, can be observed after 1 day of development. Cellular degeneration in two different pathological phenotypes—the occluded blastulae and the occluded prism—is present after 48 h of development. Results show that a greater effect on cell death is induced by the small oligomers stabilized under physiological conditions than at acid pH. In this case only occluded blastulae are found after 48 h of development.—Carrotta, R., Di Carlo, M., Manno, M., Montana, G., Picone, P., Romancino, D., San Biagio, P. L. Toxicity of recombinant β‐amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus. FASEB J. 20, E1301–E1308 (2006)

Insulin Promotes Survival of Amyloid-Beta Oligomers Neuroblastoma Damaged Cells via Caspase 9 Inhibition and Hsp70 Upregulation

Alzheimers disease (AD) and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta) in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

Large size fibrillar bundles of the Alzheimer amyloid β-protein

Self-assembly of amyloid β-protein (Aβ) and its deposition into senile plaques are distinctive features of Alzheimer’s disease. Aβ forms typical linear aggregates known as amyloid fibrils, with a diameter of a few tens of nanometers and a length spanning from hundreds of nanometers to micrometers. Fibrils eventually assemble into large size clusters and precipitate in vivo in the brain deposits. Here, we study the late stage of aggregation of Aβ(1–40) in vitro at pH 3.1. We characterize the structure of fibrillar aggregates by a combined use of different experimental techniques. Small angle light scattering, heterodyne near field scattering, large angle light scattering, ultra small angle X-ray scattering and small angle X-ray scattering measurements have been performed to highlight the structural features of amyloid bundles over several lengthscales, from nanometers to tens of micrometers. Phase contrast optical microscopy has been used to complement scattering measurements and directly visualize some morphological details. We show that elongated fibrils of Aβ with a diameter of a few nanometers are packed into large size compact bundles having a typical size of tens of micrometers. The linear morphology of fibrils is reflected in the elongated shape of bundles.

Entrapment of A?1?40 peptide in unstructured aggregates

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis.

Oligomerizing ability of newly made human Hsp60 with its mitochondrial import signal

It is currently accepted that the human Hsp60 resides and works not only in the mitochondria, the canonical residence, but also outside it. It is also known that Hsp60 although coded by a nuclear gene is synthesized in the cytosol and includes an N-terminal mitochondrial import signal (MIS), which directs the polypeptide toward the inside of the organelle where the MIS is removed. Therefore, there are at least two functional types of Hsp60, with and without MIS, the former in the cytosol the latter inside the mitochondria. A key question is: how do these two forms of Hsp60 differ beyond the fact that while one has MIS the other lacks it? How presence or absence of MIS affects the ability of Hsp60 to form oligomers, which are considered important for chaperoning peptides and assist them to reach a native state? We report here our initial observations on this issue. Typically, in the mitochondria, Hsp60 forms ring-shaped heptamers, two of which associate to build a barrel-shaped tetradecamer, the functional chaperoning complex. It is not known if the cytosolic Hsp60 with its MIS, also forms hepta- and tetradecamers. A clarification of this issue will most likely shed light on the physiological functions of extramitochondrial Hsp60, and also on its pathogenic role in Hsp60 chaperonopathies. Consequently, we compared recombinant Hsp60 bearing the MIS with the prokaryotic ortholog GroEL, which under normal conditions forms functional double-ring tetradecamers. Characteristic hydrodynamic sizes of the oligomeric complex for both systems were investigated by small angle X-ray (SAXS) and static and dynamic light scattering (SLS and DLS) in solution under similar physicochemical conditions. High Performance Liquid Chromatography (HPLC) and blue native polyacrylamide-gel electrophoresis were used to further clarify the equilibrium between the different oligomeric species of the two proteins over a wide range of concentrations. Hsp60 with MIS formed hepta- and tetradecamers similarly to GroEL. Oligomerization was dependent on concentration for GroEL and Hsp60, but for the latter, formation of larger oligomers, e.g., tetradecamers, required higher concentrations than the former.

Thermal broadening of Lb band of “trehalose coated” tyrosine and phenylalanine

We studied the thermal broadening of Lb band of tyrosine and phenylalanine embedded in a trehalose matrix. Aim of this work is to obtain information on the effects of “trehalose coating” on the coupling of the electronic transition to low frequency modes in the surrounding of the chromophore. The results obtained for the two molecular complexes put in evidence that O-H groups are involved in blocking these structures within the solid trehalose matrix and shed light on the role played by hydrogen bonds on the interactions that keep “trehalose coated” proteins rigid and solid-like.