In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-␤ (A␤) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid A␤ peptide (A␤ 1-42 ) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic A␤ 1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu 2ϩ and specific "aging" of the A␤ 1-42 solution. Maximal COX inhibition occurred when using A␤ 1-42 solutions aged for 3-6 h at 30°C. The level of A␤ 1-42 -mediated COX inhibition increased with aging time up to ϳ6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric A␤ as the only A␤ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant A␤ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous A␤ is associated with brain mitochondria and that A␤ 1-42 , possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu 2ϩ . We conclude that Cu 2ϩ -dependent A␤-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease.

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Accumulation of neurotoxic amyloid‐β (Aβ) is central to the pathology of Alzheimer’s disease (AD). Elucidating the mechanisms of Aβ accumulation will therefore expedite the development of Aβ‐targeting AD therapeutics. We examined activity of an Aβ‐degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Aβ accumulation by altering Aβ sensitivity to proteolytic degradation. An Aβ amino acid mutation found in familial AD, Aβ interactions with zinc (Zn), and increased Aβ hydrophobicity all strongly prevented Aβ degradation. Consistent to all of these factors is the promotion of specific Aβ aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Aβ accumulation by preventing normal protease activity. Zn also prevented Aβ degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn‐induced Aβ amyloid with the metal‐protein attenuating compound clioquinol reversed amyloid formation and restored the peptide’s sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Aβ‐metal interactions can inhibit Aβ accumulation by restoring the catalytic potential of Aβ‐degrading proteases.

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By altering key amino acid residues of the Alzheimer's disease-associated amyloid-b peptide, we investigated the mechanism through which amyloid-b inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-b inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-b ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-b oligomeric species. Although the amyloid-bmediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu 2+ , it was not supported by monovalent Cu + , and experiments with catalase and H 2 O 2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-b-mediated H 2 O 2 production. We propose that amyloid-b-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu 2+ , and that it may involve the formation of a redox active amyloid-b-methionine radical.

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Cullin RING E3 Ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 Ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs) and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contribute to polyubiquitylation but do not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

SummarySheep which had been either previously infected with O. circumcincta or maintained worm-free, were surgically prepared with separated fundic pouches and abomasal cannulae and subsequently infected with 20000 O. circumcincta larvae three times weekly.A reduction in food intake and increases in total acid output from the pouches and plasma pepsinogen levels were evident in both groups of sheep 4 days after repeated infections commenced; effects which increased in severity after 12 or more days. Except for a transient period of slight failure, previously infected sheep retained the capacity to acidify their abomasal contents whereas previously worm-free sheep lost this capacity. These changes were reversed between 2 and 7 days after treatment with thiabendazole (88 mg.kg−1).Secretory capacity of the fundic pouches was tested with histamine (40 μg.kg−1), the histamine antagonist (burimamide 8mg.kg−1) and atropine (100 μg kg−1). Ostertagiasis reduced or abolished the stimulatory effects of histamine. An increase in secretion volume and acid output was obtained after food was freshly provided, even though as little as 25 gm was consumed. Atropine and burimamide both caused a profound decrease in pouch secretion and acid output.These data are consistent with the hypothesis previously stated that in ostertagiasis the hypersecretion from fundic pouches is due to increased levels of circulating gastrin.