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Amyloid-beta (Aβ) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aβ oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aβ autocleavage during the early phase of aggregation, producing a typical Aβ fragment signature and that truncated Aβ peptides can form stable oligomeric complexes with full-length Aβ peptide. We show that the use of novel anti-Aβ antibodies raised against these truncated Aβ isoforms allows for monitoring and targeting the accumulation of truncated Aβ fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aβ cleavage is a highly relevant clinical feature of AD., (Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

The development of novel therapies against neurodegenerative disorders requires the ability to detect their early, presymptomatic manifestations in order to enable treatment before irreversible cellular damage occurs. Precocious signs indicative of neurodegeneration include characteristic changes in certain protein levels, which can be used as diagnostic biomarkers when they can be detected in fluids such as blood plasma or cerebrospinal fluid. In the case of synucleinopathies, cerebrospinal alpha-synuclein (α-syn) has attracted great interest as a potential biomarker; however, there is ongoing debate regarding the association between cerebrospinal α-syn levels and neurodegeneration in Parkinson disease and synucleinopathies. Post-translational modifications (PTMs) have emerged as important determinants of α-syn's physiological and pathological functions. Several PTMs are enriched within Lewy bodies and exist at higher levels in α-synucleinopathy brains, suggesting that certain modified forms of α-syn might be more relevant biomarkers than the total α-syn levels. However, the quantification of PTMs in bodily fluids poses several challenges. This review describes the limitations of current immunoassay-based α-syn quantification methods and highlights how these limitations can be overcome using novel mass-spectrometry-based assays. In addition, we describe how advances in chemical synthesis, which have enabled the preparation of α-syn proteins that are site-specifically modified at single or multiple residues, can facilitate the development of more accurate assays for detecting and quantifying α-syn PTMs in health and disease.

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity.