Figure 1.
Screening of a human brain cDNA library expressed on M13 phage.
A. Amino acid sequence of the insert of the positive phage clone designated C2. B. BLASTP analysis results. Amino acid sequence of the insert of C2 is identical to a fragment of CcOX1. C. Complete amino acid sequence of CcOX1. Residues of the insert of C2 are in bold. D. Analysis of interaction of Aβ1–42/Aβ 1–40 with C2 phage bearing the fragment of CcOX 1. Phage concentration used was 1011 per ml, and 100 µl were added to each well. M13 phage and a non-related peptide (NRP) were used as negative controls. OD at 405 was registered. Data are means of three independent experiments.
Figure 2.
Western blot analysis of recombinant C2 phage expressing the fragment of CcOX1.
1011 phage particles diluted in loading buffer were resolved on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature and immunoblotted for detection with anti-pIII antibody. Wild-type M13 phage was used as a control. Migration of the molecular mass standards as well as pIII and pIII–CcOX1 fusion protein are indicated by arrowheads.
Figure 3.
CcOX1 fragment-bearing phage bound to amyloid deposits in AD brain.
C2 phage bound to amyloid aggregates in 50 µm-thick brain tissue sections of temporal cortex from AD patients (A and B). A control non-related phage (3.15) did not bind to amyloid aggregates present on brain sections (C and D). Each panel shows, from the left: the reactivity of phage (green); the reactivity of Thiazin red (TR) or BAM 90.1, an anti-Aβ1–42 monoclonal antibody, used as positive controls to detect amyloid aggregates (red); the merge between red and green channels. A–B: scale bar represents 10 µm; C–D: scale bar represents 20 µm.
Figure 4.
CcOX1 fragment-bearing phage bound to amyloid deposits in AD brain.
C2 phage bound to amyloid aggregates in 50 µm-thick brain tissue sections of temporal cortex from AD patients (A–D) and from cognitively normally aging (NA) elderly subjects (E). Each panel shows, from the left: the reactivity of phage (green); the reactivity of BAM 90.1, an anti-Aβ1–42 monoclonal antibody (red); the merge between red and green channels. A, C and E: scale bar represents 150 µm; B and D: scale bar represents 10 µm.
Figure 5.
Co-immunoprecipitation of CcOX1 and Aβ42 from mitochondrial protein extracts from IMR-32 cells.
Aβ42 was co-incubated with mitochondrial protein lysate from IMR-32 cells, then Aβ42 and CcOX1 were immunoprecipitated using goat anti-CcOX1 antibodies or non-related goat-anti-EGF antibodies, followed by western blotting for Aβ42 and CcOX1. BAM90.1, a mouse monoclonal anti-Aβ42 antibody, and rabbit anti-Aβ42 antibodies were used to detect Aβ42. Goat polyclonal anti-CcOX1 antibodies were used to detect CcOX1.
Figure 6.
Molecular Dynamics complex of Aβ1–42 (blue) and CcOX1p (gold).
Upper panel: nine pairs of carbons (spheres) interacting through van der Waals forces forming hydrophobic contacts between both peptides are showed. Insets show the hydrogen-bond (green dots) between E11-Y54 (left); the salt bridge (red dots) of H13 and H14 with D51 (left) and the salt bridge between E22-H61 (right).