Mechanism of attenuation of muscle protein degradation induced by tumor necrosis factor-alpha and angiotensin II by beta-hydroxy-beta-methylbutyrate

Both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with beta-hydroxy-beta-methylbutyrate (HMB; 50 microM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-alpha/IFN-gamma and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRDelta6, in response to TNF-alpha/IFN-gamma, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and -8. HMB also attenuated activation of PKR, suggesting that it was important in protein degradation. Formation of ROS was attenuated by rotenone, an inhibitor of the mitochondrial electron transport chain, nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, and SB 203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), which also attenuated total protein degradation. Activation of p38 MAPK by PKR provides the link to ROS formation. These results suggest that TNF-alpha/IFN-gamma and ANG II induce muscle protein degradation by a common signaling pathway, which is attenuated by HMB, and that this involves the initial activation of caspase-3 and -8, followed by autophosphorylation and activation of PKR, which then leads to increased ROS formation via activation of p38 MAPK. Increased ROS formation is known to induce protein degradation through the ubiquitin-proteasome pathway.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

TATPred:a Bayesian method for the identification of twin arginine translocation pathway signal sequences

The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name. There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor. The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.

Validation of protein carbonyl measurement:a multi-centre study

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15 min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.