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Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

ABSTRACT: PrP 106-126 conserves the pathogenic and physicochemical properties of the Scrapie isoform of the prion protein. PrP 106-126 and other amyloidal proteins are capable of inducing ion permeability through cell membranes, and this property may represent the common primary mechanism of pathogenesis in the amyloid-related degenerative diseases. However, for many amyloidal proteins, despite numerous phenomenological observations of their interactions with membranes, it has been difficult to determine the molecular mechanisms by which the proteins cause ion permeability. One approach that has not been undertaken is the kinetic study of protein-membrane interactions. We found that the reaction time constant of the interaction between PrP 106-126 and membranes is suitable for such studies. The kinetic experiment with giant lipid vesicles showed that the membrane area first increased by peptide binding but then decreased. The membrane area decrease was coincidental with appearance of extramembranous aggregates including lipid molecules. Sometimes, the membrane area would increase again followed by another decrease. The kinetic experiment with small vesicles was monitored by circular dichroism for peptide conformation changes. The results are consistent with a molecular simulation following a simple set of well-defined rules. We deduced that at the molecular level the formation of peptide amyloids incorporated lipid molecules as part of the aggregates. Most importantly the amyloid aggregates desorbed from the lipid bilayer, consistent with the macroscopic phenomena observed with giant vesicles. Thus we conclude that the main effect of membrane-mediated amyloid formation is extraction of lipid molecules from the membrane. We discuss the likelihood of this effect on membrane ion permeability.

SUBMITTER: Sun Y

PROVIDER: S-EPMC4554819 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

Sun Yen Y Hung Wei-Chin WC Lee Ming-Tao MT Huang Huey W HW

Biochimica et biophysica acta 20150726 10 Pt A

PrP 106-126 conserves the pathogenic and physicochemical properties of the Scrapie isoform of the prion protein. PrP 106-126 and other amyloidal proteins are capable of inducing ion permeability through cell membranes, and this property may represent the common primary mechanism of pathogenesis in the amyloid-related degenerative diseases. However, for many amyloidal proteins, despite numerous phenomenological observations of their interactions with membranes, it has been difficult to determine ...[more]

PMID: 26215743

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Project description:Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP) fragment 106-126 (PrP(106-126)). We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126) in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126)-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb). The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126). The results showed that PrP(106-126) treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α), increased iNOS expression and nitric oxide (NO) production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126)-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126)-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126)-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126). Together, these results suggest that CD36 is involved in PrP(106-126)-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126) and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides and open perspectives for new therapeutic strategies for prion diseases by modulation of CD36 signaling.

Project description:Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc). The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues. We recently showed that a synthetic peptide corresponding to residues 106-126 [PrP-(106-126)] of the human PrP is toxic to neurons and trophic to astrocytes in vitro. Our experiments were aimed at verifying whether PrP-(106-126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann-Sträussler-Scheinker disease-namely PrP-(89-106), PrP-(106-114), PrP-(127-147)-were capable of stimulating circulating leucocytes. Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies. PrP-(106-126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2-. production in monocytes and neutrophils. Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106-126) to the cell suspension in the concentration range 5-50 microM. The increase in intracellular Ca2+ elicited by PrP-(106-126) was dose-dependent in the range 5-500 microM. PrP-(106-126) stimulated O2-. production in monocytes and neutrophils in a dose- (10-300 microM) and time-(5-30 min) dependent manner in the presence of 10 microM dihydrocytochalasin B. Both the increase in Ca2+ concentration and the O2-. production were partially sensitive to pertussis toxin. PrP-(106-126) stimulated leucocyte migration in a dose-dependent (30-300 microM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.

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Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

Publications

Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

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