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首页> 外文期刊>Journal of Cachexia, Sarcopenia and Muscle >Proteasomal activity-based probes mark protein homeostasis in muscles
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Proteasomal activity-based probes mark protein homeostasis in muscles

机译：基于蛋白酶体活性的探针标记肌肉中的蛋白质稳态

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Abstract Background Protein homeostasis, primarily regulated by the ubiquitin?￠????proteasome system is crucial for proper function of cells. In tissues of post-mitotic cells, the impaired ubiquitin?￠????proteasome system is found in a wide range of neuromuscular disorders. Activity-based probes (ABPs) measure proteasomal proteolytic subunits and can be used to report protein homeostasis. Despite the crucial role of the proteasome in neuromuscular pathologies, ABPs were not employed in muscle cells and tissues, and measurement of proteasomal activity was carried out in vitro using low-throughput procedures. Methods We screened six ABPs for specific application in muscle cell culture using high throughput call-based imaging procedures. We then determined an in situ proteasomal activity in myofibers of muscle cryosections. Results We demonstrate that LWA300, a pan-reactive proteasomal probe, is most suitable to report proteasomal activity in muscle cells using cell-based bio-imaging. We found that proteasomal activity is two-fold and three-fold enhanced in fused muscle cell culture compared with non-fused cells. Moreover, we found that proteasomal activity can discriminate between muscles. Across muscles, a relative higher proteasomal activity was found in hybrid myofibers whereas fast-twitch myofibers displayed lower activity. Conclusions Our study demonstrates that proteasomal activity differ between muscles and between myofiber types. We suggest that ABPs can be used to report disease progression and treatment efficacy.
机译：背景技术主要由泛素蛋白酶体系统调节的蛋白质稳态对细胞的正常功能至关重要。在有丝分裂后细胞的组织中，广泛的神经肌肉疾病中发现泛素蛋白酶体系统受损。基于活动的探针（ABP）可测量蛋白酶体的蛋白水解亚基，可用于报告蛋白质稳态。尽管蛋白酶体在神经肌肉病理学中起着至关重要的作用，但ABP并未在肌肉细胞和组织中使用，并且使用低通量方法在体外进行蛋白酶体活性的测量。方法我们使用高通量基于呼叫的成像程序筛选了六种ABP在肌肉细胞培养中的特定应用。然后，我们确定了肌肉冰冻切片肌纤维中的原位蛋白酶体活性。结果我们证明，泛反应性蛋白酶体探针LWA300最适合使用基于细胞的生物成像技术报告肌细胞中的蛋白酶体活性。我们发现，与非融合细胞相比，融合肌肉细胞培养中蛋白酶体活性提高了两倍和三倍。此外，我们发现蛋白酶体活性可以区分肌肉。在整个肌肉中，混合肌纤维中蛋白酶体的活性相对较高，而快速抽搐的肌纤维中蛋白酶体的活性较低。结论我们的研究表明，蛋白酶的活性在肌肉之间和肌纤维类型之间是不同的。我们建议ABP可用于报告疾病进展和治疗效果。

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