Department of Human Genetics, LUMC, Leiden, The Netherlands.
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden, The Netherlands.
J Cachexia Sarcopenia Muscle. 2017 Oct;8(5):798-807. doi: 10.1002/jcsm.12211. Epub 2017 Jul 3.
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.
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.
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.
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.
蛋白质稳态主要受泛素-蛋白酶体系统调控，对于细胞的正常功能至关重要。在后有丝分裂细胞的组织中，广泛存在于多种神经肌肉疾病中的泛素-蛋白酶体系统受损。活性基探针（ABP）可测量蛋白酶体的蛋白酶亚基，并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 ABPs 并未在肌肉细胞和组织中使用，并且蛋白酶体活性的测量是在体外使用低通量程序进行的。
我们使用高通量基于细胞的成像程序筛选了六种适用于肌肉细胞培养的 ABP，然后确定了肌肉冷冻切片中肌纤维的原位蛋白酶体活性。
我们证明，LWA300 是一种广谱的蛋白酶体探针，最适合使用基于细胞的生物成像来报告肌肉细胞中的蛋白酶体活性。我们发现，与未融合的细胞相比，融合的肌肉细胞培养物中的蛋白酶体活性增强了两倍和三倍。此外，我们发现蛋白酶体活性可以区分肌肉。在肌肉之间，杂种肌纤维中的蛋白酶体活性相对较高，而快肌纤维的活性较低。
我们的研究表明，蛋白酶体活性在肌肉之间以及肌纤维类型之间存在差异。我们建议 ABPs 可用于报告疾病进展和治疗效果。
