Monitoring the Process of HypF Fibrillization and Liposome Permeabilization by Protofibrils

Relini, Annalisa; Torrassa, Silvia; Rolandi, R.; Gliozzi, Alessandra; Rosano, Camillo; Canale, Claudio; Bolognesi, Martino; Plakoutsi, Georgia; Bucciantini, Monica; Chiti, Fabrizio; Stefani, Massimo

doi:10.1016/j.jmb.2004.03.054

Cited by 97 publications

(99 citation statements)

References 58 publications

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“…We measured calcein release from unilamellar vesicles made of neutral and negatively charged phospholipids as an index of membrane permeability, as described previously (16). After 12 h of aggregation, A␤py3-42 caused a 23% increase of membrane permeability, which instead was only slightly altered by A␤1-42 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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β-Amyloid Is Different in Normal Aging and in Alzheimer Disease

Piccini

Russo

Gliozzi

et al. 2005

Journal of Biological Chemistry

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186

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The mechanism of neurodegeneration caused by ␤-amyloid in Alzheimer disease is controversial. Neuronal toxicity is exerted mostly by various species of soluble ␤-amyloid oligomers that differ in their N-and C-terminal domains. However, abundant accumulation of ␤-amyloid also occurs in the brains of cognitively normal elderly people, in the absence of obvious neuronal dysfunction. We postulated that neuronal toxicity depends on the molecular composition, rather than the amount, of the soluble ␤-amyloid oligomers. Here we show that soluble ␤-amyloid aggregates that accumulate in Alzheimer disease are different from those of normal aging in regard to the composition as well as the aggregation and toxicity properties.A series of evidence indicates that progressive cerebral accumulation of ␤-amyloid (A␤), 2 a proteolytic product of transmembrane protein APP, is the primary pathogenic event of Alzheimer disease (AD) (1). Recent clues indicate that small, soluble A␤ aggregates produce more severe synaptic dysfunction and neuronal damage than do A␤ polymers (2-5). This behavior is common to all known pathogenic and nonpathogenic amyloidogenic peptides (6, 7). Soluble A␤ is detectable early in the cerebral cortex of subjects at risk for AD pathology, several years before the formation and deposition of amyloid fibrils (8). Hence, the analysis of soluble A␤ in brain tissue allows the characterization of the toxic form of the peptide.A strong argument against the amyloid hypothesis is the abundant and constant deposition of A␤ in the brains of elderly subjects, in the absence of signs of neuronal degeneration and dementia (9 -11). The reasons for the absence of pathogenic effect exerted by A␤ in normal aging are unknown. The issue has important therapeutic implications, because the major strategies to prevent and cure AD are focused on halting A␤ accumulation (12).In brains from Alzheimer disease (AD) and Down syndrome patients, three major species of soluble A␤ have been identified by mass spectrometry: the full-length form, A␤1-42, which has a relative molecular mass of 4.5 kDa, and two N-terminal peptides truncated at residue 3 (A␤3-42) and residue 11 (A␤11-42) with relative molecular masses of 4.2 and 3.5 kDa, respectively (13, 14). The 4.2-and 3.5-kDa bands are more prominent in familial AD carrying presenilin 1 mutations than in sporadic AD, suggesting that the ratio of soluble A␤ species may dictate the toxicity of the aggregates (15).We predicted that the composition of soluble A␤ underlies the different effect exerted by the molecule in AD and in normal aging. To investigate this hypothesis, we studied the composition and properties of aggregation and toxicity as well as the damage produced on artificial membranes of soluble A␤, comparing these areas in sporadic AD and cognitively normal elderly subjects with abundant amyloid plaques in cerebral cortex. MATERIALS AND METHODSTissues-We used frozen blocks and formalin-fixed sections of frontal cortex from 14 cases with late onset sporadic AD (mean age at death 80 Ϯ ...

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Section: Resultsmentioning

confidence: 99%

“…Membrane Permeability Assay-Vesicle leakage induced by A␤ peptides was evaluated by means of the release of calcein (Sigma) as described previously (16). A␤ aggregates, obtained at different incubation times, were added at a final concentration of 1.5 M to a phospholipid vesicles suspension (lipid concentration 0.05 mM).…”

Section: Methodsmentioning

confidence: 99%

β-Amyloid Is Different in Normal Aging and in Alzheimer Disease

Piccini

Russo

Gliozzi

et al. 2005

Journal of Biological Chemistry

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186

157

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“…First, it forms fibrils that are morphologically, tinctorially and structurally similar to those found in amyloid deposition diseases (Chiti et al, 2001;Relini et al, 2004). Second, HypF-N forms pre-fibrillar oligomers that are toxic to cells in culture and in animal models (Bucciantini et al, 2002;Bucciantini et al, 2004;Cecchi et al, 2005;Baglioni et al, 2006;Pellistri et al, 2008).…”

Section: Introductionmentioning

confidence: 87%

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Cappelli

Penco²,

Mannini³

et al. 2016

Biological Chemistry

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. (2016). Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations. Biological Chemistry: official scientific journal of the GBM, 397 (5), 401-415. Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations AbstractLiving systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsCappelli, S., Penco, A., Mannini, B., Cascella, R., Wilson, M. R., Ecroyd, H., Li, X., Buxbaum, J. N., Dobson, C. M., Cecchi, C., Relini, A. & Chiti, F. (2016 measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasises the efficiency and versatility of these protein molecules.

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“…HypF-N has been found to be able to convert in vitro, under appropriate conditions, into fibrils that are morphologically and tinctorially indistinguishable from those associated with disease (35,36). The fibril formation process of HypF-N consists of a number of steps during which pre-fibrillar aggregates precede formation of structured fibrillar species (4,36). In this paper we describe how the expression of a range of mutants of this protein in the cytosol of E. coli cells has provided us with an opportunity to explore the aggregation of a protein within the living organism in which it is naturally expressed.…”

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confidence: 99%

Investigating the Effects of Mutations on Protein Aggregation in the Cell

Calloni

Zoffoli

Stefani

et al. 2005

Journal of Biological Chemistry

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The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

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Monitoring the Process of HypF Fibrillization and Liposome Permeabilization by Protofibrils

Cited by 97 publications

References 58 publications

β-Amyloid Is Different in Normal Aging and in Alzheimer Disease

β-Amyloid Is Different in Normal Aging and in Alzheimer Disease

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Investigating the Effects of Mutations on Protein Aggregation in the Cell

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