Rate Of Total Protein Degradation Research Articles

XIAP Reduces Muscle Proteolysis Induced by CKD

X-chromosome-linked inhibitor of apoptosis protein (XIAP) is an endogenous caspase inhibitor. Caspase-3 contributes to the muscle wasting associated with chronic kidney disease (CKD) and other systemic illnesses, but whether XIAP modulates muscle wasting in CKD is unknown. Here, overexpression of XIAP in cultured skeletal muscle cells decreased protein degradation induced by serum deprivation, suggesting that caspase-mediated proteolysis contributes to muscle atrophy. We generated transgenic mice that overexpress human XIAP specifically in skeletal muscle (mXIAP) and evaluated muscle protein degradation induced by CKD. mXIAP mice with normal kidney function exhibited mild skeletal muscle hypertrophy. Muscle weights of mXIAP mice with CKD (mXIAP-CKD) were indistinguishable from wild-type mice, suggesting that overexpression of XIAP in skeletal muscle protects from CKD-induced muscle atrophy. The rate of total protein degradation, proteasome chymotrypsin-like activity, and caspase-3-mediated actin cleavage all were lower in muscle isolated from mXIAP-CKD mice compared with wild-type CKD mice. Concomitant with the reduction in overall proteolysis, mRNA levels of ubiquitin, muscle-specific ring finger 1, and atrogin-1/muscle atrophy F-box were lower in mXIAP-CKD mice, suggesting that decreased expression of the ubiquitin-proteasome pathway components may contribute to the protein-sparing effects of XIAP. In summary, these results demonstrate that XIAP inhibits multiple aspects of protein degradation in skeletal muscle during CKD.

A reexamination of the effect of exercise on rate of muscle protein degradation.

The purpose of this study was to examine the effect of exercise on the rate of protein degradation in rat skeletal muscle. The rates of total and myofibrillar protein degradation were determined by the measurement of the rates of release of tyrosine and 3-methylhistidine, respectively, from the perfused single rat leg. This method measures the rate of protein degradation in the entire lower leg and does not suffer from the limitations inherent in methods that rely on urinary excretion. The rate of total protein degradation was increased by exercise and involved increased flux through the lysosomal pathway, while the breakdown of myofibrillar protein was unchanged. The changes in the rates of protein degradation during the recovery period were greatly influenced by energy intake. Again the rate of myofibrillar protein degradation was unchanged or slightly increased during the recovery period, after either level or downgrade running. Exercise did prevent the increase in the rate of total protein degradation caused by food restriction, which may have important implications in weight reduction diets.

ß2-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of β2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective β2-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10−5 m clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10−5 m ICI 118·551, a selective β2-antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10−4 m dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by β2-adrenoceptors, with the participation of a cAMP-dependent pathway.

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells.

The rates of [3H]N(tau)-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [3H]methionine, were used to measure myofibrillar protein degradation. In fused C2C12 myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions +/- leucine, but in the absence of [3H]-methionine, rates of protein accretion and synthesis over 24-48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types.

A reexamination of the effect of exercise on rate of muscle protein degradation

The purpose of this study was to examine the effect of exercise on the rate of protein degradation in rat skeletal muscle. The rates of total and myofibrillar protein degradation were determined by the measurement of the rates of release of tyrosine and 3-methylhistidine, respectively, from the perfused single rat leg. This method measures the rate of protein degradation in the entire lower leg and does not suffer from the limitations inherent in methods that rely on urinary excretion. The rate of total protein degradation was increased by exercise and involved increased flux through the lysosomal pathway, while the breakdown of myofibrillar protein was unchanged. The changes in the rates of protein degradation during the recovery period were greatly influenced by energy intake. Again the rate of myofibrillar protein degradation was unchanged or slightly increased during the recovery period, after either level or downgrade running. Exercise did prevent the increase in the rate of total protein degradation caused by food restriction, which may have important implications in weight reduction diets.

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.

Effects of food deprivation and refeeding on total protein and actomyosin degradation

Total protein and actomyosin degradation rates were determined in perfused rat hemicorpus preparations. By simultaneously measuring the release of two nonmetabolizable amino acids phenylalanine and N tau-methylhistidine from the hemicorpus, the respective rates of total protein and actomyosin degradation could be calculated. When rats were deprived of food for 48 h, the rate of total protein degradation increased to 148% of the fed controls. If rats were food deprived and then refed for 24 h, the degradation rate decreased to only 79% of the rate of fed controls. Measurement of N tau-methylhistidine release indicated that food deprivation led to a dramatic increase in the rate of actomyosin degradation (427% of fed), whereas refeeding decreased the actomyosin degradation rate to that of the fed controls. Calculations of the fractional degradation rates show that actomyosin breaks down at a much slower rate than the nonactomyosin proteins (1.5 vs. 20.8%/day in preparations from fed rats, and 6.2 vs. 28.2%/day in preparations from food-deprived rats). Therefore, the contribution of actomyosin breakdown to total muscle protein breakdown is small in the fed state (11%) and increased threefold after food deprivation. The addition of insulin to the perfusion medium decreased the rate of total protein degradation by 18% in preparations from food-deprived rats with no significant effect on actomyosin degradation. Thus, in vitro, insulin's major effect may be to decrease the degradation of more rapidly turning over, nonactomyosin proteins. Protein degradation, as well as protein synthesis, contributes to the adaptation of muscle to starvation and refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Interference with lysosomal proteolysis fails to reduce cardiac myosin degradation

Chloroquine (a lysosomotropic agent) and leupeptin (an inhibitor of thiol proteinases including lysosomal cathepsins B, H, and L) reduced the rate of total protein degradation in cultured fetal mouse hearts by 25% (p < 0.01). Simultaneously, the rate of degradation of myosin was not diminished by either agent. In contrast, insulin reduced the rate of total protein degradation by 16% and of myosin degradation by 13% (p < 0.01 for both). These results indicate that primary interference with lysosomal proteolytic processes fails to inhibit myosin degradation; it is suggested that the degradation of myosin (and perhaps other myofibrillar proteins) is normally accomplished via nonlysosomal mechanisms.

Suppression of myocardial protein degradation in the rat during fasting. Effects of insulin, glucose, and leucine.

To study the effects of leucine, glucose, and insulin on myocardial protein degradation in fed and fasted nutritional states, we developed and validated a sensitive method for measuring rates of total protein degradation in rat isolated left atrial preparations. Fasting resulted in a progressive decrease in myocardial protein breakdown to 71% of control over a 24-hour period, with no further reduction in degradation rate between 24 and 72 hours of fasting. Insulin (100 mU/ml) suppressed atrial protein degradation by 38% in fed animals (P less than 0.001) and by 51% in fasted animals (P less than 0.001). Glucose alone had no effect on protein degradation in either nutritional state. At 5 times normal plasma levels, leucine suppressed protein breakdown by 21% in fed and by 15% in fasted animals. The decrease in degradation induced by fasting and the absence of an effect of glucose are in contrast to the behavior reported for skeletal muscle.

Inhibition of sporulation in the water mold Blastocladiella emersonii by antipain

Blastocladiella emersonii express two different types of caseinolytic activities during the process of sporulation. They can be distinguished in vitro on the basis of their sensitivity to antipain. The alkaline protease activity is inhibited by antipain and PMSF, whereas the second enzyme, denoted here as the caseinolytic activity, is not inhibited by antipain but is sensitive to PMSF and concanavalin A. In vivo, antipain blocks sporulation when added to cultures during the first 60 min of sporulation, but if added 90 min after sporulation is induced, it is biologically ineffective. In both cases, antipain enters the cells and decreases the rate of total protein degradation by 60%. The antisporulation effect of antipain cannot be reversed by washing the cells. The ability of cells which have been pretreated with antipain to sporulate can be recovered, but only after a period of growth. These data provide evidence for the critical role of the alkaline protease for a limited period of time during the initial phases of sporulation in Blastocladiella. A hypothesis based on the processing of preformed proteins by the alkaline protease as a key control mechanism for sporulation is presented.