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Several approaches have been used in an effort to identify proteins that interact with β‐amyloid precursor protein (APP). However, few studies have addressed the identification of proteins associated with APP in brain tissue from patients with Alzheimer's disease. We report the results of a pilot proteomic study performed on complexes immunoprecipitated with APP in brain samples of patients with Alzheimer's disease and normal control subjects. The 21 proteins identified could be grouped into five functional classes: molecular chaperones, cytoskeletal and structural proteins, proteins involved in trafficking, adaptors, and enzymes. Among the proteins identified, six had been reported previously as direct, indirect, or genetically inferred APP interactors. The other 15 proteins immunoprecipitated with APP were novel potential partners. We confirmed the APP interaction by Western blotting and coimmunolocalization in brain tissues, for 5 of the 21 interactors. In agreement with previous studies, our results are compatible with an involvement of APP in axonal transport and vesicular trafficking, and with a potential association of APP with cellular protein folding/protein degradation systems. Ann Neurol 2005;58:277–289

The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with α-catenin, a component of the adhesion complex. Spectrin (αIIβII) and α-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and α-catenin in vitro. By surface plasmon resonance and in vitrobinding assays, we find that α-catenin binds αIIβII spectrin with an apparent Kdof ≈20–100 nm. By gel-overlay assay, α-catenin binds recombinant βII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of α-catenin is fully accounted for in recombinant peptides encompassing the NH2-terminal 228 amino acid region of α-catenin. Anin vivorole for the interaction of spectrin with α-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective α-catenin. Transfection of these cells with wild-type α-catenin reestablishes α-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between α-catenin and the amino-terminal domain of β-spectrin augment the interaction between α-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has become a fundamental tool for the identification and analysis of peptides and proteins. MALDI-TOF is well suited for the analysis of complex biological mixtures because samples are crystallized onto a solid support that can be washed to remove contaminants and salts prior to laser desorption. A number of approaches for immobilizing samples onto MALDI targets have been put forth. These include the use of different chemical matrices and the immobilization of samples onto different solid supports. In large part though, the preparation of MALDI targets has been an empirical exercise that often requires a unique series of conditions for every sample. Here, a simple method for the application of peptide mixtures onto MALDI targets is put forth. This method differs because peptides are added directly to a sample of nitrocellulose dissolved in acetone, allowing them to interact in solution-phase organic solvent. This solution-phase mixture is then spotted to the MALDI target and evaporated, forming a homogenous solid surface for laser desorption. This procedure is robust, highly sensitive, tolerant to detergents, and easily learned. In our hands, the method provides as much as a 10-fold enhancement to the detection of tryptic peptide fragments derived from in-gel digests. Copyright 2000 Academic Press.

Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between φT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 μmfor pT5; in contrast, dissociation constants for calmodulin were greater than 6 μmfor all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.