Polymerization of amyloid ␤-peptide (A␤) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease. Here, we show that peptides incorporating a short A␤ fragment (KLVFF; A␤ 16 -20 ) can bind full-length A␤ and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys 16 , Leu 17 , and Phe 20 are critical for binding to A␤ and inhibition of A␤ fibril formation. A mutant A␤ molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues A␤ 16 -20 serve as a binding sequence during A␤ polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and nonpeptide agents aimed at inhibiting A␤ amyloidogenesis in vivo.The preeminent neuropathological feature of Alzheimer's disease is the deposition of amyloid in the brain parenchyma and cerebrovasculature (1, 2). The basic components of the amyloid are thin fibrils of a peptide termed A␤ (3, 4). This peptide is a 40-to 42-amino acid-long proteolytic fragment of the Alzheimer amyloid precursor protein (APP), 1 a protein expressed in most tissues (5). Genetic and neuropathological studies provide strong evidence for a central role of A␤ amyloid in the pathogenesis of Alzheimer's disease (6), but the pathophysiological consequences of the amyloid deposition are unclear. However, it has been suggested that A␤ polymers and amyloid are toxic to neurons, either directly or via induction of radicals, and hence cause neurodegeneration (7-9). Previous studies indicate that A␤ polymerization in vivo and in vitro is a specific process that probably involves interactions between binding sequences in the A␤ peptide (10 -12). A rational pharmacological approach for prevention of amyloid formation would therefore be to use drugs that specifically interfere with A␤-A␤ interaction and polymerization. We hypothesized that ligands capable of binding to and blocking such sequences might inhibit amyloid fibril formation as outlined schematically in Fig. 1. Our strategy in searching for an A␤ ligand was to identify binding sequences in A␤ and then, based on their primary structures, synthesize a peptide ligand. Binding sequences were identified by systematically synthesizing short peptides corresponding to sequences of the A␤ molecule. The minimum length of an identified binding sequence was determined by truncating the peptide. Residues critical for binding were identified by alanine scanning. These critical residues were then substituted in an A␤ fragment (A␤ ) that normally is capable of forming amyloid fibrils (13,14) in order to determine if they indeed are important for A␤ amyloid fibril formation. Finally, it was determined if the identified ligand, in addition to binding to the A␤ molecule, was capable of inhibiting fibril formation of A␤ . EXPERIMENTAL PROCEDURES Materials-Synthetic A␤1-40 and all other soluble peptides were synthesized b...

Polymerization of the amyloid beta (A␤) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected A␤-fragments containing the A␤ 16 -20 sequence, previously shown essential for A␤-A␤ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was A␤ 14 -23 . Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from A␤ 1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of A␤ 14 -23 oligomers in an antiparallel ␤-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of A␤-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

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In Alzheimer's disease and spongiform encephalopathies proteins transform from their native states into fibrils. We find that several amyloid-forming proteins harbor an ␣-helix in a polypeptide segment that should form a ␤-strand according to secondary structure predictions. In 1324 nonredundant protein structures, 37 ␤-strands with >7 residues were predicted in segments where the experimentally determined structures show helices. These discordances include the prion protein

Most endocrine hormones are produced in tissues and organs with permeable microvessels that may provide an excess of hormones to be transported by the blood circulation to the distal target organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence of other angiogenic factors. We provide evidence that leptininduced new blood vessels are fenestrated. Under physiological conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient ob͞ob mice. Thus, leptin plays a critical role in the maintenance and regulation of vascular fenestrations in the adipose tissue. Leptin induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate that leptin has another new function-the increase of vascular permeability.leptin ͉ obesity ͉ vascular fenestrations ͉ neovascularization

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The aneurysm wall covered with thrombus is thinner and shows more frequent signs of inflammation, apoptosis of SMCs, and degraded extracellular matrix. These findings suggest that thrombus formation and accumulation of inflammatory cells may perturb the structural integrity and stability of the vessel wall and thereby increase the risk for aneurysm rupture.

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.