ABSTRACT
It has been shown recently that the generation of an abnormal transmembrane form of the prion protein ((Ctm)PrP) is involved in the neurodegeneration process during inherited and infectious prion diseases but a causative relationship has never been established. We wanted to know if and how the proposed transmembrane domain of PrP could induce neuronal dysfunction. Thus, we investigated the neurotoxic properties of two peptides whose sequences are encompassed within this domain. We show that PrP peptides 118-135 and 105-132 as well as an amidated more soluble peptide 105-132 induce the death of pure cortical neurons originating from normal and PrP knockout mice. This can be correlated with the high propensity of these peptides to insert stably into and to destabilize cell membranes. Through this study, we have identified a novel mechanism of neurotoxicity for PrP, which directly involves membrane perturbation; this mechanism is independent of fibril formation and probably corresponds to the effect of the transmembrane insertion of (Ctm)PrP.
Subject(s)
Neurons/drug effects , Peptide Fragments/toxicity , Prion Diseases/metabolism , Prions/toxicity , Amyloid beta-Peptides/toxicity , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Excitatory Amino Acids/pharmacology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Models, Molecular , Monte Carlo Method , Neurons/metabolism , Neurons/pathology , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Peptide Fragments/ultrastructure , Prion Diseases/etiology , Prions/biosynthesis , Prions/chemical synthesis , Prions/chemistry , Prions/ultrastructure , Protein Structure, TertiaryABSTRACT
The interaction between porcine pancreatic phospholipase A2 and a homogeneous population of micelles of the subtrate analogue n-hexadecylphosphorylcholine containing 155 lipid monomers was studied by light scattering, equilibrium gel filtration, and isothermal calorimetry. From the detergent/protein molar ratio and the equivalent "molecular weight" of the resulting complex it is concluded that insertion of the enzyme into the detergent micelle results in a protein--detergent complex containing two phospholipase A2 molecules and 80 lipid monomers at 25 degrees C. The affinity constants and complex composition have been determined at different temperatures, allowing calculation of the thermodynamic parameters of the binding process. It is concluded that the interaction of phospholipase A2 with micellar lipids is predominantly hydrophobic.
Subject(s)
Colloids , Micelles , Phospholipases , Animals , Kinetics , Mathematics , Pancreas/enzymology , Protein Binding , Swine , ThermodynamicsABSTRACT
Dielectric measurements were made on aqueous solutions of native BSA monomers at three concentrations and at about eighty separate frequencies in the range 0.03-800 MHz. All measurements were made at 25 degrees C on solutions of pH = 5.0, this being near the isoelectric point. The analysis shows the presence of a double component beta dispersion at frequencies below 10 MHz plus two delta dispersions with relaxation frequencies of around 15 MHz and 100 MHz. The origin at a molecular level of the beta and the higher frequency delta dispersion can be established with confidence but the remaining delta dispersion cannot be interpreted unambiguously. With the inclusion of the well known gamma dispersion at frequencies in excess of 1 GHz the measurements indicate the existence of five clearly separated dispersion regions. This is one more dispersion than previously suggested in the literature for a protein solution of this nature.
