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The tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The type of tau isoforms (4R/3R) observed in brain aggregates is used to classify tauopathies. However, distinguishing tauopathies in cerebrospinal fluid remains challenging. Here, we demonstrated that the difference in tau isoforms between tauopathies is only observed in aggregates, not in soluble brain extracts. We therefore used untargeted mass spectrometry to characterize all post-translational modifications of both the aggregated and the soluble tau protein obtained from human brain tissue of patients with Alzheimer’s disease, cortico-basal degeneration, Pick’s disease, and fronto-temporal lobe degeneration. We found specific soluble signatures predictive of each tauopathy and its peculiar type of aggregated tau isoforms. These findings provide potential targets for developing cerebrospinal fluid assays able to distinguish between tauopathies in vivo. The comparison between soluble and aggregated tau features indicates potential mechanisms of tau aggregation, providing novel therapeutic leads for these diseases.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified pep-tide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-␣-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features , and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to–α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

International audience; Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns.

International audience; Recent studies have evidenced that oxidatively damaged DNA, which potentially leads to carcinogenic mutations and aging, may result from the direct absorption of low-energy photons (4250 nm). Herein, the primary species, i.e., ejected electrons and base radicals associated with such damage in duplexes with an alternating guanine-cytosine sequence are quantified by nanosecond transient absorption spectroscopy. The one-photon ionization quantum yield at 266 nm is 1.2 Â 10 À3 , which is similar to those reported previously for adenine-thymine duplexes. This means that the simple presence of guanine, the nucleobase with the lowest ionization potential, does not affect photo-ionization. The transient species detected after 3 ms are identified as deprotonated guanine radicals, which decay with a half-time of 2.5 ms. Spectral assignment is made with the help of quantum chemistry calculations (TD-DFT), which for the first time, provide reference absorption spectra for guanine radicals in duplexes. In addition, our computed spectra predict the changes in transient absorption expected for hole localization as well as deprotonation (to cytosine and bulk water) and hydration of the radical cation.

International audience; Telomeres, which are involved in cell division, carcinogenesis, and aging and constitute important therapeutic targets, are prone to oxidative damage. This propensity has been correlated with the presence of guanine-rich sequences, capable of forming four-stranded DNA structures (G-quadruplexes). Here, we present the first study on oxidative damage of human telomere G-quadruplexes without mediation of external molecules. Our investigation has been performed for G-quadruplexes formed by folding of GGG(TTAGGG)3 single strands in buffered solutions containing Na+ cations (TEL21/Na+). Associating nanosecond time-resolved spectroscopy and quantum mechanical calculations (TD-DFT), it focuses on the primary species, ejected electrons and guanine radicals, generated upon absorption of UV radiation directly by TEL21/Na+. We show that, at 266 nm, corresponding to an energy significantly lower than the guanine ionization potential, the one-photon ionization quantum yield is 4.5 × 10–3. This value is comparable to that of cyclobutane thymine dimers (the major UV-induced lesions) in genomic DNA; the quantum yield of these dimers in TEL21/Na+ is found to be (1.1 ± 0.1) × 10–3. The fate of guanine radicals, generated in equivalent concentration with that of ejected electrons, is followed over 5 orders of magnitude of time. Such a quantitative approach reveals that an important part of radical cation population survives up to a few milliseconds, whereas radical cations produced by chemical oxidants in various DNA systems are known to deprotonate, at most, within a few microseconds. Under the same experimental conditions, neither one-photon ionization nor long-lived radical cations are detected for the telomere repeat TTAGGG in single-stranded configuration, showing that secondary structure plays a key role in these processes. Finally, two types of deprotonated radicals are identified: on the one hand, (G-H2)• radicals, stable at early times, and on the other hand, (G-H1)• radicals, appearing within a few milliseconds and decaying with a time constant of ∼50 ms.