Atrogin1/MAFbx is an ubiquitin ligase that mediates muscle atrophy in a variety of catabolic states. We recently found that H2O2 stimulates atrogin1/MAFbx gene expression. Since the cytokine tumor necrosis factor-alpha (TNF-alpha) stimulates both reactive oxygen production and general activity of the ubiquitin conjugating pathway, we hypothesized that TNF-alpha would also increase atrogin1/MAFbx gene expression. As with H2O2, we found that TNF-alpha exposure up-regulates atrogin1/MAFbx mRNA within 2 h in C2C12 myotubes. Intraperitoneal injection of TNF-alpha increased atrogin1/MAFbx mRNA in skeletal muscle of adult mice within 4 h. Exposing myotubes to either TNF-alpha or H2O2 also produced general activation of the mitogen-activated protein kinases (MAPKs): p38, ERK1/2, and JNK. The increase in atrogin1/MAFbx gene expression induced by TNF-alpha was not altered significantly by ERK inhibitor PD98059 or the JNK inhibitor SP600125. In contrast, atrogin1/MAFbx up-regulation and the associated increase in ubiquitin conjugating activity were both blunted by p38 inhibitors, either SB203580 or curcumin. These data suggest that TNF-alpha acts via p38 to increase atrogin1/MAFbx gene expression in skeletal muscle.

. Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes. Am J Physiol Cell Physiol 285: C806-C812, 2003. First published May 28, 2003 10.1152/ajpcell.00129.2003.-Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 M H2O2 increased the rate of 125 I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system. cachexia; proteolysis; reactive oxygen species; free radicals; aging ELEVATED LEVELS of reactive oxygen species (ROS) have been linked to the muscle atrophy that occurs in various inflammatory diseases (24). Animal studies by Buck and Chojkier (3) support the concept that elevated ROS levels might promote muscle protein loss. These investigators observed that antioxidants, either D-␣-tocopherol or BW775c, administered to mice can attenuate the muscle atrophy induced by tumor necrosis factor (TNF)-␣, a cytokine that stimulates mitochondrial production of ROS (8,15,26). At the cellular level, a link between ROS and muscle protein loss was recently demonstrated by Gomes-Marcondes and Tisdale (7). They showed that incubation of C 2 C 12 myotubes with 100 M H 2 O 2 for 24 h stimulates protein degradation and increases expression of the 20S proteasome ␣-subunit and E2 14K , a ubiquitin carrier protein. These findings suggest that ROS stimulate loss of muscle protein by upregulating elements of the ubiquitin-proteasome pathway.In this pathway, ubiquitin conjugation to a protein substrate initiates degradation of the protein by the 26S proteasome complex. The ubiquitin-activating enzyme (E1 protein) activates ubiquitin, which is then transferred to a ubiquitin carrier protein (E2). The E2 protein interacts with a ubiquitin ligase (E3) to catalyze transfer of ubiquitin to a protein substrate, marking the substrate for proteasomal degradation as ubiquitin accumulates. Specificity of the system is attributed to the E2 and E3 proteins that target particular proteins for degradation (13). For example, in skeletal muscle E2 14K and E3␣ work together to transfer ubiquitin to protein substrates that contain basic and large hydrophobic NH 2 -t...

Bacteria, exemplified by enteropathogenic Escherichia coli (E. coli), rely on elaborate acid resistance systems to survive acidic environment (such as the stomach). Comprehensive understanding of bacterial acid resistance is important for prevention and clinical treatment. In this study, we report a previously uncharacterized type of acid resistance system in E. coli that relies on L-glutamine (Gln), one of the most abundant food-borne free amino acids. Upon uptake into E. coli, Gln is converted to L-glutamate (Glu) by the acid-activated glutaminase YbaS, with concomitant release of gaseous ammonia. The free ammonia neutralizes proton, resulting in elevated intracellular pH under acidic environment. We show that YbaS and the amino acid antiporter GadC, which exchanges extracellular Gln with intracellular Glu, together constitute an acid resistance system that is sufficient for E. coli survival under extremely acidic environment.

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally-engineered PMTs to label substrates of the corresponding native PMTs. Here 4-azido-but-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e. g. EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the non-histone targets of EuHMT1/2. Comparison of EuHMT1/2’s methylomes indicates that the two human PMTs, though similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.