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%

Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

Two-thirds of the lipid A in wild-type-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH 4 VO 3 -treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

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.

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.