Blondelle Jordan, Shapiro Paige, Domenighetti Andrea A, Lange Stephan
Division of Cardiology, University of California San Diego, La Jolla, CA-92093 USA.
Rehabilitation Institute of Chicago, Chicago, IL-60611 USA; Department of Physical Medicine and Rehabilitation, Northwestern University, Chicago, IL-60611, USA.
J Mol Biol. 2017 Apr 7;429(7):1045-1066. doi: 10.1016/j.jmb.2017.02.012. Epub 2017 Feb 24.
The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.
泛素连接酶E3在肌肉稳态中的作用在肌肉萎缩过程中最为人所知，因为泛素连接酶E3的底物衔接蛋白atrogin-1是最具特征的肌肉萎缩相关蛋白之一。我们研究了在体外成肌细胞终末分化以及乙酰胆碱受体聚集以建立神经肌肉接头的过程中，泛素连接酶E3的活性是否也至关重要。泛素连接酶E3的活性通过与类泛素小分子修饰蛋白nedd8的翻译后修饰来调节。在C2C12成肌细胞以及来自小鼠和人类的原代卫星细胞分化的早期或晚期，使用Nae1抑制剂MLN4924（pevonedistat）或针对nedd8的小干扰RNA，我们发现泛素连接酶E3的活性在体外肌肉细胞分化程序的每一步都是必需的。我们进一步研究了已知的终末肌肉分化转录抑制因子，即锌指蛋白38（ZBTB38）、含碱性螺旋-环-螺旋结构域蛋白41（Bhlhe41）和DNA结合抑制因子1（Id1）。由于它们在终末肌肉分化中已确定的作用，我们推测这些潜在的泛素连接酶E3底物的积累可能部分导致了所观察到的表型。MLN4924目前正在癌症患者中进行临床试验，我们的实验突出了对这些经常出现恶病质的患者肌肉稳态和再生能力的担忧。
