Cerebral amyloid angiopathy (CAA) due to ␤-amyloid (A␤) is one of the specific pathological features of familial Alzheimer's disease. A␤ mainly consisting of 40-and 42-mer peptides (A␤40 and A␤42) exhibits neurotoxicity and aggregative abilities. All of the variants of A␤40 and A␤42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the A␤40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type A␤40. Similar tendency was observed for A␤42 mutants at positions 22 and 23 whose neurotoxicity was 50 -200 times stronger than that of the corresponding A␤40 mutants, suggesting that these A␤42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-A␤42 and D23N-A␤42 was similar to that of wild-type A␤42, E22Q-A␤42 and E22K-A␤42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of A␤ mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that ␤-sheet content of the A␤ mutants correlates with their aggregation. However, ␤-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue ␤-turn significantly enhanced the aggregative ability. Alzheimer's disease (AD)1 is neuropathologically characterized by the progressive deposition of amyloid in the brain parenchyma and cortical blood vessels (1). This deposition mainly consists of 40-and 42-mer ␤-amyloid peptides (A␤40 and A␤42) generated from amyloid precursor protein by two proteases, ␤-and ␥-secretases (2, 3). Cerebral amyloid angiopathy (CAA) in familial Alzheimer's disease is linked to missense mutations inside the A␤-coding region in the amyloid precursor protein. The mutations of A␤ sequence are concentrated at positions 21-23 and are called Flemish (A21G) (4), Arctic (E22G) (5, 6), Dutch (E22Q) (7), Italian (E22K) (8), and Iowa (D23N) (9) mutations. These A␤ mutant peptides may play a pathological role in the CAAs because wild-type A␤ peptides induce neuronal death in vitro (10). Neurotoxicity and formation of amyloid fibrils of some CAA-related A␤40 mutants have been independently reported by several groups (11-15). However, there are no reports on the neurotoxicity and aggregation of the CAA-related A␤42 mutants with the exception of Dutch mutation (E22Q) (11), the investigation of which is essential to reveal the mechanism of CAA because wild-type A␤42 shows considerably stronger neurotoxicity and aggregative ability than wild-type A␤40 (11). Moreover, it is indispensable to simultaneously compare neurotoxicity and aggregative ability of all of the CAA-related A␤40 and A␤42 mutants in the same conditions such as pH, peptide concentration, reaction buffer, and temperature.It is difficult to synthesize A␤42 with 14 hydrophobic and/or ...

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The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.ubiquitin-binding protein | ubiquitin ligase | ubiquitination

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