Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide-bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, 13C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N-and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the ␣-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N-and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N-and O-sulfo groups such as that found in the liver and the brain. Human apolipoprotein E (apoE)1 is a 299-residue polymorphic protein that facilitates the transport and metabolism of lipids (1). ApoE is a ligand for members of the low density lipoprotein (LDL) receptor family, heparin, and heparan sulfate proteoglycans (HSPGs) (2, 3). It is composed of two domains: a 22-kDa NH 2 -terminal domain (residues 1-191) and a 10-kDa COOH-terminal domain (residues 216 -299) (4). The 22-kDa NH 2 -terminal domain contains the primary HSPGbinding site (residues 140 -150) (5) colocalized with the LDL receptor binding site (6 -8).Binding of apoE to HSPG is an initial step in the localization of apoE-containing lipoproteins to the surface of several different types of cells (9). After localization, the apoE-containing lipoproteins are transported into the cell by pathways dependent on either the LDL receptor or the LDL receptor-related protein (LRP) or by direct uptake of an apoE-containing lipoprotein-HSPG complex (10). Binding of apoE to HSPG affects neurite extension in neurons (11, 12) and localizes secreted apoE to the surface of macrophages (13). Binding of apoE to HSPG may also play a role in Alzheimer's disease through either competition between apoE and the amyloid precursor protein (APP) for HSPG-binding sites or by modulation of the HSPG/LRP uptake pathway (14 -17).The best understood physiological role of the binding of apoE to HSPG is in lipoprotein remnant clearance....

Alzheimer's disease-associated amyloid-β (Aβ) peptide is neurotoxic as an oligomer, but not as a monomer, by an unknown mechanism. We showed previously that Aβ interacts with the amyloid-β precursor protein (AβPP), leading to caspase cleavage and cell death induction. To characterize this structure and interaction further, we purified the extracellular domain of AβPP695 (eAβPP) and its complex with Aβ oligomers (AβOs) of varying sizes, and then performed small angle X-ray scattering (SAXS). In the absence of any Aβ, eAβPP was a compact homodimer with a tight association between the E1 and E2 domains. Dimeric Aβ oligomers induced monomerization of eAβPP while larger oligomers also bound eAβPP but preserved the homodimer. Efficient binding of the larger oligomers correlated with the presence of prefibrillar oligomers, suggesting that the eAβPP binding is limited to a conformational subset of Aβ oligomers. Both forms of Aβ bound to eAβPP at the Aβ-cognate region and induced dissociation of the E1 and E2 domains. Our data provide the first structural evidence for Aβ-AβPP binding and suggest a mechanism for differential modulation of AβPP processing and cell death signaling by Aβ dimers versus conformationally-specific larger oligomers.

A systematic approach was used to identify AβPP-selective BACE inhibitors (ASBI) and to evaluate their in vivo ability to modulate AβPP processing selectively. We identified a bioflavonoid nutritional supplement as a molecular lead that acts as an ASBI in cell models, and show that increasing brain levels of this bioflavonoid through a pro-drug approach leads to reduction of Aβ42 in an Alzheimer’s disease mouse model. ASBIs represent a novel class of candidate therapeutic agents for Alzheimer’s disease.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

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In the version of this article initially published, the discovery and synthesis of the dafachronic acids was not attributed properly in two instances. On p. 892, the statement "the DAF-9 cytochrome P450 completes the synthesis of the dafachronic-acid hormones 11,13 " should have cited references 11, 13 and 14, and the statement "DAF-9 catalyzes two sequential oxidation steps at the C26 position to generate metabolites that are first hydroxylated and then carboxylated to products named Δ 4 -and Δ 7 -dafachronic acid, respectively 13 " should have cited reference 14 instead of 13. The errors have been corrected in the PDF and HTML versions of this article. In the version of this article initially published, the authors concluded, on the basis of the substantial (approximately five-fold) N1 regioselectivity observed for reactions that form the unsubstituted triazole ureas shown in Figures 1-3, that the major regioisomeric product for the 4-substituted triazole ureas shown in Figures 1 and 3 was also the N1 regioisomer. They have since determined by X-ray crystallography (provided as Supplementary Data Sets 1 and 2) that the N2 regioisomer is the major product for the 4-substituted triazole ureas. The structures have been corrected in the HTML and PDF versions of the article and in the chemical probe