Effects on the ubiquitin proteasome system after closed soft-tissue trauma in rat skeletal muscle

Abstract

Previous studies have suggested that an increased catabolic stage of skeletal muscle in pathological situations is mainly a reflection of ubiquitin-proteasome system-controlled proteolysis. The proteolytic mechanisms that occur after local muscle trauma are poorly defined. We investigated the effects of closed soft-tissue trauma on ubiquitin-proteasome dependent protein breakdown in rats (n = 25). The enzymatic activities of the ubiquitination and proteasome reactions were both reduced (p < 0.05) immediately after contusion of the hind limb musculus extensor digitorum longus. The same effect was observed in extracts of lung tissue from the injured animals. Cellular levels of free and protein-conjugated ubiquitin were significantly elevated upon decreased proteolytic activity. Our data support an early-state anti-proteolytic role of the ubiquitin-proteasome pathway after local injury. This further implies that there is a yet-to-be elucidated complex regulatory mechanism of muscle regeneration that involves various proteolytic systems.

Motor protein function in skeletal abdominal muscle of cachectic cancer patients

“…This was also confirmed by our quantitative immunoblot protein analysis approach using an adequate calibration technique 45 . Our values of ∼200 ng/50 μg total protein for dimers and ∼400 ng/50 μg for monomers are well in the range for values of 2–3 μg free ubiquitin per mg total protein given in a recent rat muscle trauma model 58 . Thus, we are confident that our technique faithfully tracks ubiquitin protein multimers in skeletal human muscle.…”

Tourniquet-induced ischaemia during total knee arthroplasty results in higher proteolytic activities within vastus medialis cells: a randomized clinical trial

“…The principal findings of the present study were that the proteasome-dependent and proteasome-independent peptidase activities were statistically significantly higher during tourniquet-induced 60-min ischaemia as compared to the non-tourniquet group, suggesting an activation of the molecular cascade of muscle protein degradation, which might explain muscle atrophy after TKA. These results were based on a human model of skeletal muscle ischaemia and did not find to be meaningfully comparable to the previously published data about directly traumatized human [30] and/or rat skeletal muscle [27]. This can mainly be explained due to differences in the study design and experimental setting.…”

“…The present study included irreversible selective proteasome inhibitors such as epoxomicin and ADA. For the purpose of quantification of proteasomedependent/proteasome-independent peptidase activities, the application of epoxomicin and ADA found to be reliable [15,26,27,30]. The high regulation of cytosolic Ub is considered essential for the cell metabolism [1,7,24].…”

“…Each of the mixtures was incubated for 3-min time intervals at 37 °C, containing 30 µg muscle tissue extract, assay buffer (50 mM Tris/HCL, 5 mM MgCl, 1 mM dithioerythritol; PH 8.0), 1 mM ATP and 0.2 µg/µl biotinylated ubiquitin. The reaction was terminated by adding a sample buffer (Lämmli) and separated by SDS-PAGE [27]. The test-retest reliability was >0.95.…”

“…In order to differentiate between proteasome-dependent and proteasome-independent peptidase activities, proteasome inhibitors such as epoxomicin or adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-methyl-sulphone were added to the reaction mixture for the assessment of chymotryptic-, tryptic-and caspase-like peptidase activities, respectively. The results based on the following equations [27]: Proteasome-dependent and proteasome-independent proteolytic activities were calculated as pkat/mg total protein from the mean of three identical tests. The test-retest reliability was >0.95.…”