Proximity-dependent biotin identification links cholesterol catabolism with branched-chain amino acid degradation in Mycobacterium smegmatis.

Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA-based proximity-dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA-HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein-protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG_1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched-chain amino acids. As cholesterol and branched-chain amino acid catabolism both generate propionyl-CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl-CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG_1634 and MSMEG_6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched-chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein-protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets.

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition.

BACKGROUND: DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood. METHODS AND RESULTS: Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation. CONCLUSIONS: The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication. GENERAL SIGNIFICANCE: Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.

Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters.

Older age is one of the strongest risk factors for severe COVID-19. In this study, we determined whether age-associated cellular senescence contributes to the severity of experimental COVID-19. Aged golden hamsters accumulate senescent cells in the lungs, and the senolytic drug ABT-263, a BCL-2 inhibitor, depletes these cells at baseline and during SARS-CoV-2 infection. Relative to young hamsters, aged hamsters had a greater viral load during the acute phase of infection and displayed higher levels of sequelae during the post-acute phase. Early treatment with ABT-263 lowered pulmonary viral load in aged (but not young) animals, an effect associated with lower expression of ACE2, the receptor for SARS-CoV-2. ABT-263 treatment also led to lower pulmonary and systemic levels of senescence-associated secretory phenotype factors and to amelioration of early and late lung disease. These data demonstrate the causative role of age-associated pre-existing senescent cells on COVID-19 severity and have clear clinical relevance.

Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription.

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Mitochondrial activity as an indicator of fish freshness.

The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨm) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨm and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness.

Short-term exposure to gold nanoparticle suspension impairs swimming behavior in a widespread calanoid copepod.

Calanoid copepods play an important role in the functioning of marine and brackish ecosystems. Information is scarce on the behavioral toxicity of engineered nanoparticles to these abundant planktonic organisms. We assessed the effects of short-term exposure to nonfunctionalized gold nanoparticles on the swimming behavior of the widespread estuarine copepod Eurytemora affinis. By means of three-dimensional particle tracking velocimetry, we reconstructed the trajectories of males, ovigerous and non-ovigerous females. We quantified changes in their swimming activity and in the kinematics and geometrical properties of their motion, three important descriptors of the motility patterns of zooplankters. In females, exposure to gold nanoparticles in suspension (11.4 µg L-1) for 30 min caused depressed activity and lower velocity and acceleration, whereas the same exposure caused minimal effects in males. This response differs clearly from the hyperactive behavior that is commonly observed in zooplankters exposed to pollutants, and from the generally lower sensitivity of female copepods to toxicants. Accumulation of gold nanoparticles on the external appendages was not observed, precluding mechanical effects. Only very few nanoparticles appeared sporadically in the inner part of the gut in some samples, either as aggregates or as isolated nanoparticles, which does not suggest systemic toxicity resulting from pronounced ingestion. Hence, the precise mechanisms underlying the behavioral toxicity observed here remain to be elucidated. These results demonstrate that gold nanoparticles can induce marked behavioral alterations at very low concentration and short exposure duration. They illustrate the applicability of swimming behavior as a suitable and sensitive endpoint for investigating the toxicity of nanomaterials present in estuarine and marine environments. Changes in swimming behavior may impair the ability of planktonic copepods to interact with their environment and with other organisms, with possible impacts on population dynamics and community structure.