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Fig. 4 | Alzheimer's Research & Therapy

Fig. 4

From: Non-canonical soluble amyloid-beta aggregates and plaque buffering: controversies and future directions for target discovery in Alzheimer’s disease

Fig. 4

Cysteine cleavage of AD patient-derived Aβ aggregates and predicted structural models of Aβ aggregates. a Primary sequence analysis of Aβ1-42. Cysteine, proline, threonine, and tryptophan are not present within the 42 residues. b Protein fragmentation using 2-nitro-5-thiocyanatobenzoic acid (NTCB). In this study, proteins were pre-treated with 5 mM dithiothreitol (DTT) for 30 minutes at room temperature (RT) to reduce cysteine thiols. NTCB at final concentration of 5 mM was then added to cyanylate proteins at cysteine residues for 4 h. After cyanlyation, 2 mM NaOH was used to adjust pH to 9, resulting in cleavage N-terminal to cysteines. The reaction was allowed to proceed for 16 h at RT. The NTCB fragmented proteins were then used for downstream applications directly. (i) SDS-page gel analysis of reduced NTCB fragmented proteins. The positive control bovine serum albumin (BSA) and immunoglobulin G (IgG) proteins known to contain cysteine residues were fragmented by NTCB successfully, as indicated by the change from non-cleaved (NC) to cleaved (CL). The negative control synthetic monomeric Aβ1-42 was not fragmented by NTCB due to the absence of cysteine. The majority of proteins in AD patient-derived brain lysate were successfully fragmented by NTCB. (ii) Western blot analysis of synthetic Aβ1-42 before and after digestion showed no significant difference in size or band intensity. c Size exclusion chromatography profiling of total protein and soluble Aβ aggregates from an AD patient using Superdex 200 column. AD patient-derived brain lysate (1 mL) was separated by Superdex 200 column. An ELISA-based soluble Aβ aggregate assay [23] was then used to assess for soluble Aβ aggregates in each fraction. High molecular weight soluble Aβ aggregates were detected in fractions 7 to 10, with the estimated size larger than 670 kDa. d Size exclusion chromatography and soluble Aβ aggregate assay profiling of DTT and NTCB alone. DTT and NTCB were added to sample buffer and incubated as described above. DTT and NTCB (1 mL) in sample buffer without any protein was separated using a Superdex 200 column. DTT and NTCB were distributed from fractions 20 to 25, consistent with low molecular weight chemicals (molecular weights of DTT and NTCB are 154.25 and 224.19 g/mol, respectively). AβS26C dimer standard was then added to each fraction at a final concentration of 400 pg/mL. There was loss of Aβ dimer standard signal in fractions 20 to 25 but no effect on other fractions. e Simplified models of Aβ aggregates and potential outcomes after DTT and NTCB treatment. Model (i): more than one Aβ monomer binds to a protein complex specifically or nonspecifically. In this case, after a successful cleavage N-terminal to cysteine residues with NTCB, the protein complex core will be fragmented into numerous small protein fragments and peptides with no more than one Aβ monomer attached to each fragment/peptide. This would result in loss of signal on the soluble Aβ aggregate assay. Model (ii): one or more low molecular weight Aβ aggregates attach to a protein complex core specifically or nonspecifically, similar to model (i). However, after successful cleavage of cysteine using NTCB, low molecular weight Aβ aggregates attached to smaller protein fragments or peptides can still be detected by the soluble Aβ aggregate assay. Therefore, lower molecular weight Aβ aggregates are expected after a NTCB treatment in model (ii). Model (iii): numerous Aβ monomers aggregate and form a high molecular weight Aβ complex without other proteins. In this case, Aβ aggregates will not be fragmented by NTCB due to the absence of cysteine residues. Therefore, Aβ aggregates will show no change before and after NTCB treatment. f Size exclusion chromatography profiling of AD patient-derived soluble Aβ aggregates before and after DTT and NTCB treatment, and predicted structure of AD patient-derived soluble Aβ aggregates. (i) AD patient-derived Aβ aggregate were treated with DTT alone or with DTT followed by NTCB as described above. As shown in (i), DTT alone has no detectable effect on soluble Aβ aggregates; after the treatment with DTT and NTCB, however, the soluble Aβ aggregate assay detected a change in the size distribution of the soluble Aβ aggregates ranging from fraction 7 (>670 kDa) to fraction 16 (approximately 44 kDa). There was a potential false positive signal in fractions 21 to 23 as described above for d. The partial cleavage of AD patient-derived soluble Aβ aggregates is most consistent with a more complex structure compared with the three simplified models described in e. (ii) Hypothetical structure of soluble Aβ aggregates. After DTT and NTCB treatment, high molecular weight soluble Aβ aggregates appear to have been partially fragmented into various sizes of smaller Aβ aggregates, indicating that proteins with cysteine residues are present in at least some of the soluble Aβ aggregates. The detectable smaller aggregates could contain various types of non-cleavable high molecular weight proteins (or protein fragments) with more than one Aβ monomer attached, or the smaller aggregates could be pure Aβ. Importantly, we cannot determine the extent to which the DTT and NTCB fully cleaved all cysteine-containing proteins. Thus, the continued detection of some high molecular weight soluble Aβ aggregates does not necessarily indicate that these species consist only of Aβ. (Original figure: H. Jiang and D. Brody)

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