Reinvestigation of the chemical structure of (-amyloid nonpathological by-product of cellular metabolism, whereas A3-(1-42) may have the more important role in the formation of neuritic plaques.With this in mind, we reexamined amyloid from the cerebrovasculature of AD brains and found that A,B-(1-42) is also the major form in these deposits. Interestingly, the amount of racemization and isomerization at aspartyl residues is much less than in neuritic plaque Af-(1-42). The localization of these undegradable aggregates suggests that their deposition might be linked to a compromised blood-brain barrier. MATERIALS AND METHODSHuman brains obtained at autopsy met the diagnostic criteria for AD established by the National Institutes of Health Neuropathology Panel (10) and by the Consortium to Establish a Registry for AD (CERAD) (11). Morphometric analyses identified brains that contained large amounts of AP in compact cores and in the blood vessels. Left hemispheres were analyzed histopathologically, while right hemispheres were stored at -70°C for subsequent A,3 isolation.Purification of A,B from Leptomeningeal Blood Vessels. The leptomeninges were gently pulled from the surface of 1-cmthick coronal sections with the aid of a dissecting microscope and immersed in 0.1 M Tris HCl (pH 8.0; TB) at 4°C. Blood vessels larger than 1 mm in diameter were discarded, and the remaining tissue was cut with scissors into 1-to 2-mm pieces. The tissue was washed eight times with 1 liter of TB at 4°C with continuous stirring for 10 min and was collected by filtration (45-,um mesh). After resuspension of this material in 20 vol of 2 mM CaCl2 in TB, 0.3 mg of collagenase (Worthington) and 10 p.g of DNase I (Worthington) were added per ml, and the suspension was shaken for 18 hr at 37°C. Large debris was removed by filtration (350-,gm mesh), and the smaller insoluble material was recovered by centrifugation at 6000 x g for 15 min. The resulting pellet was resuspended in 100 vol of 2% SDS in TB and incubated for 2 hr at room temperature, after which the insoluble material was again recovered by centrifugation (see above) and washed twice with distilled water. The pellet was then dissolved with 8 vol of 98% (vol/vol) glass-distilled formic acid (15 min at room temperature) and centrifuged at 430,000x g for 15 min in Polyallomer tubes in a TLA 100.2 rotor (Beckman). Pure A8 was isolated from the clear supernatant by size-exclusion chromatography with a Superose 12 column (10 x 300 mm) on a Pharmacia-LKB fast protein liquid chromatography (FPLC) system with a running buffer of 75%

In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of A␤ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic A␤-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8 -10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. Although evidence implicates ␤-amyloid peptide (A␤) in the pathogenesis of Alzheimer's disease (AD) 1 (reviewed in Ref. 1), little is known about the nature of the A␤ mediating the pathology. Toxicity initially was attributed to aggregated A␤ in amyloid plaques (1), the morphological hallmarks of AD brains. A␤-(1-42) is the major peptide constituent of amyloid plaques (2), and increased production of the 42-amino acid peptide correlates with an earlier onset of AD (1). However, recent studies show that small quantities of A␤-(1-42) also exists as soluble peptide in the plasma, cerebrospinal fluid, and cerebral cortex of AD and normal individuals and are also secreted by cells in tissue culture (3-13). Utilizing ultracentrifugation, graded membrane filtration, and ELISA, we have recently isolated and quantitated the oligomeric water-soluble A␤ present in the brains of AD and control individuals (13). The levels of insoluble A␤ in AD brains are at least 100 times higher than those found in control brains. The amounts of water-soluble A␤ in AD brains are approximately six times higher than those detected in control brains. Interestingly, we isolated an A␤ fraction, from the A␤ water-soluble oligomeric pool, with a molecular mass of Ͻ10 kDa containing monomeric and/or dimeric forms of A␤ peptide (13). In all probability these peptides represent the initial building blocks that may ultimately aggregate into insoluble A␤ filaments. In the course of analyzing the chemical composition of AD neuritic plaque and vascular amyloid, we have purified stable dimeric and trimeric components of A␤-(1-40/42) (2, 14 -15). In the present study we report the chemical and morphological characteristics of the dimeric A␤ as elucidated by atomic force microscopy and transmission electron microscopy techniques. In addition, the potential for toxicity of the AD brain-derived A␤-(1-40/42) dimer was assessed on glial-neuronal cell cultures. MATERIALS AND METHODSPurification of A␤-(1-42) from AD Brain-Brains were obtained from eight patients who died of AD (postmortem delay 3-6 h). After separation of the leptomeninges, the right hemispheres were frozen at Ϫ70°C. Examination of the left hemispheres revealed numerous neuritic plaq...

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL)

Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.

Mouse CD1d1, a member of the CD1 family of evolutionarily conserved major histocompatibility antigen-like molecules, controls the differentiation and function of a T lymphocyte subset, NK1+ natural T cells, proposed to regulate immune responses. The CD1d1 crystal structure revealed a large hydrophobic binding site occupied by a ligand of unknown chemical nature. Mass spectrometry and metabolic radiolabeling were used to identify cellular glycosylphosphatidylinositol as a major natural ligand of CD1d1. CD1d1 bound glycosylphosphatidylinositol through its phosphatidylinositol aspect with high affinity. Glycosylphosphatidylinositol or another glycolipid could be a candidate natural ligand for CD1d1-restricted T cells.

Electrostatic interactions between a basic epitope containing adjacent arginine residues and an acidic epitope containing a phosphorylated serine are involved in receptor heteromerization. In the present study, we demonstrate that this arginine-phosphate electrostatic interaction possesses a "covalent-like" stability. Hence, these bonds can withstand fragmentation by mass spectrometric collision-induced dissociation at energies similar to those that fragment covalent bonds and they demonstrate an extremely low dissociation constant by plasmon resonance. The present work also highlights the importance of phosphorylation-dephosphorylation events in the modulation of this electrostatic attraction. Phosphorylation of the acidic epitope, a casein kinase one consensus site, makes it available to interact with the basic epitope. On the other hand, phosphorylation of serine and/or threonine residues adjacent to the basic epitope, a protein kinase A consensus site, slows down the attraction between the epitopes. Although analyzed here in the frame of receptor heteromerization, the arginine-phosphate electrostatic interaction most likely represents a general mechanism in protein-protein interactions.

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lysoGPIs is comparable with that of the intact GPIs, whereas the activity of Man 3 -GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man 3 -GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-B-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis.