The E3 ubiquitin ligase TRIM62 and inflammation-induced skeletal muscle atrophy

Introduction: Intensive care unit (ICU)-acquired weakness (ICUAW) complicates the disease course of critically ill patients. Inflammation and acute-phase response occur directly within myocytes and contribute to ICUAW. We observed that TRIM62, an E3-ubiquitin ligase and modifier of inflammation, is increased in skeletal muscle of ICUAW patients. We investigated regulation and function of muscular TRIM62 in critical illness. Methods: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores more than or equal to 8 underwent two skeletal muscle biopsies from the vastus lateralis at median days 5 and 15 in ICU. Four patients undergoing elective orthopedic surgery served as controls. TRIM62 expression and protein content was analyzed in these biopsies. Kinetics of Trim62, Atrogin1 and MuRF1 expression were determined in the gastrocnemius/plantaris and tibialis anterior from mouse models of inflammation, denervation and starvation induced muscle atrophy to differentiate between these contributors of ICUAW. Cultured myocytes were used for mechanistic analyses. Results: TRIM62 expression and protein content was increased early and remained elevated in muscle from critically ill patients. In all three animal models muscular Trim62 expression was early and continuously increased. Trim62 was expressed in myocytes and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway. Knockdown of Trim62 by siRNA inhibited lipopolysaccharide induced interleukin-6 expression. Conclusions: TRIM62 is activated in muscle of critically ill patients. It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes. Trial registration: Current Controlled Trials, ISRCTN77569430. Registered 13 February 2008

Metabolite profiling at the cellular and subcellular level reveals metabolites associated with salinity tolerance in sugar beet

Hossain MS, Persicke M, ElSayed AI, Kalinowski J, Dietz K-J. Metabolite profiling at the cellular and subcellular level reveals metabolites associated with salinity tolerance in sugar beet. Journal of Experimental Botany. 2017;68(21-22):5961-5976.Sugar beet is among the most salt-tolerant crops. This study aimed to investigate the metabolic adaptation of sugar beet to salt stress at the cellular and subcellular levels. Seedlings were grown hydroponically and subjected to stepwise increases in salt stress up to 300 mM NaCl. Highly enriched fractions of chloroplasts were obtained by nonaqueous fractionation using organic solvents. Total leaf metabolites and metabolites in chloroplasts were profiled at 3 h and 14 d after reaching the maximum salinity stress of 300 mM NaCl. Metabolite profiling by gas chromatography- mass spectrometry (GC-MS) resulted in the identification of a total of 83 metabolites in leaves and chloroplasts under control and stress conditions. There was a lower abundance of Calvin cycle metabolites under salinity whereas there was a higher abundance of oxidative pentose phosphate cycle metabolites such as 6-phosphogluconate. Accumulation of ribose-5-phosphate and ribulose-5-phosphate coincided with limitation of carbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Increases in glycolate and serine levels indicated that photorespiratory metabolism was stimulated in salt-stressed sugar beet. Compatible solutes such as proline, mannitol, and putrescine accumulated mostly outside the chloroplasts. Within the chloroplast, putrescine had the highest relative level and probably assisted in the acclimation of sugar beet to high salinity stress. The results provide new information on the contribution of chloroplasts and the extra-chloroplast space to salinity tolerance via metabolic adjustment in sugar beet

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

Chi BK, Busche T, Van Laer K, et al. Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress. Antioxidants & Redox Signaling. 2014;20(4):589-605.Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Results: Transcriptomics revealed the strong upregulation of the disulfide stress sigma(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives

Astroturf seed traps for studying hydrochory

1. Astroturf mats can effectively trap diaspores dispersed by tidal water. 2. Within four tidal inundations, up to 745 propagules per m(2) and between three and eight different species per astroturf mat were trapped. Overall, 15 different species were collected on the astroturf mats, 10 of which were also present in the local vegetation. The other five species must have come from further away, making the method also suitable for studying long-distance dispersal. 3. The seed-retaining ability of the astroturf did not differ significantly for species with different seed characteristics. 4. The method will be useful for assessing actual seed dispersal at specific sites and over specific periods of time, and has particular relevance in predicting and evaluating the success of ecological restoration projects