I. PHOTOAFFINITY CROSSLINKING OF ALZHEIMER'S DISEASE ?-AMYLOID FIBRILS II. PROTEOMIC ANALYSIS OF ENDOTHELIN-1 STIMULATED ASTROCYTES
The assembly of the ?-amyloid peptide (A?) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. This work demonstrates the novel application of photoaffinity crosslinking to determine high-resolution structural constraints on A? monomers within amyloid fibrils. Photoreactive A? 1-40 ligands were synthesized by substituting L-p-benzoyl-phenylalanine (Bpa) for phenylalanine at position 4 (A? 1-40 F4Bpa), phenylalanine at position 19 (A? 1-40 F19Bpa), or tyrosine at position 10 (A? 1-40 Y10Bpa). These peptides were incorporated into synthetic amyloid fibrils, and upon photoactivation produced crosslinked Aand? dimers. The position of photoinsertion was analyzed by proteolysis of A? dimers dissociated from the fibrils, followed by mass spectrometric analysis. MALDI-TOF mass spectrometry identified a crosslinked tryptic fragment formed by the photoinsertion of Bpa4 into the C-terminal region of A? 1-40 . MS/MS experiments and further chemical modifications of the crosslinked dimer led to the localization of the intermolecular photocrosslink between the ketone of the Bpa4 side chain and the ?-methyl group of the Met35 side chain. A? fibrils containing Bpa19-A? formed covalent crosslinked dimers, however the position of the crosslink could not be determined. Trypsin digestion of Bpa10-A? crosslinked dimers generated a crosslinked fragment in which Bpa10 photoinserts into the A?29-40 region of A? 1-40 . This finding is consistent with the intermolecular constraint produced by the Bpa4 photocrosslinking studies. Taken together these studies provide strong support for an antiparallel alignment of A? molecules as they assemble into amyloid fibrils. Reactive gliosis is an invariant feature of the central nervous system's response to injury. The hypertrophic astrocytes that form the gliotic scar influence the fate of surviving neurons through both physical and chemical interactions. To elucidate the molecular mediators of reactive gliosis, a proteomic analysis of endothelin-1 stimulated hypertrophy of cultured astrocytes was performed. Specific mediators of cytoskeletal reorganization (caldesmon, calponin, alpha B-crystallin, stathmin, CRMP-2), cell adhesion (vinculin, galectin-1), and signal transduction (RACK-1) were among the proteins differentially expressed by ET-1 treated astrocytes. The results validate the use of proteomic arrays to quantitate differential protein expression levels on a more global scale and presents potential molecular mechanisms that drive reactive hypertrophy and modulate the astrocytes' interactions with neurons following CNS injury.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:beta amyloid photoaffinity crosslinking alzheimer s disease reactive gliosis proteomics
Date of Publication:01/01/2001