J Membr Biol. 2003 May 1;193(1):35-45. Copper modulation of ion channels of PrP[106-126] mutant prion peptide fragments.
Kourie JI, Kenna BL, Tew D, Jobling MF, Curtain CC, Masters CL, Barnham KJ, Cappai R.
Membrane Transport Group, Department of Chemistry, The Faculties, The Australian National University, Canberra City, Australian Capital Territory, 0200 Australia. Joseph.Kourinu.edu.au
We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.
PMID:_12879164 [PubMed - in process]
Biochemistry. 2003 Aug 5;42(30):9022-7. Fibrillar aggregates of the tumor suppressor p53 core domain.
Departamento de Bioquimica Medica and Centro Nacional de Ressonancia Magnetica Nuclear, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.
Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.
PMID:_12885235 [PubMed - in process]
Biophys J. 2003 Aug;85(2):707-18. Theoretical modeling of prion disease incubation.
Kulkarni RV, Slepoy A, Singh RR, Cox DL, Pazmandi F.
Department of Physics, University of California, Davis, California, USA. kulkarnhysics.ucdavis.edu
We apply a theoretical aggregation model to laboratory and epidemiological prion disease incubation time data. In our model, slow growth of misfolded protein aggregates from small initial seeds controls the latent or lag phase; aggregate fissioning and subsequent spreading leads to an exponential growth phase. Our model accounts for the striking reproducibility of incubation times for high dose inoculation of lab animals. In particular, low dose yields broad incubation time distributions, and increasing dose narrows distributions and yields sharply defined onset times. We also explore how incubation time statistics depend upon aggregate morphology. We apply our model to fit the experimental dose-incubation curves for distinct strains of scrapie, and explain logarithmic variation at high dose and deviations from logarithmic behavior at low dose. We use this to make testable predictions for infectivity time-course experiments.
PMID:_12885622 [PubMed - in process]
Biophys J. 2003 Aug;85(2):1176-85. Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: insight into dynamics and properties.
Sekijima M, Motono C, Yamasaki S, Kaneko K, Akiyama Y.
Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology, 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan. sekijimbrc.jp
A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP(C)) to its pathogenic isoform (PrP(Sc)). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP(C) to PrP(Sc) and the dynamics and functions of PrP(C) remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the beta-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).
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