Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
Cytoskeleton (Hoboken). 2020 Oct;77(10):456-479. doi: 10.1002/cm.21641. Epub 2020 Nov 7.
De novo assembly of myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in premyofibrils, followed by insertions of additional proteins and increased organization in nascent myofibrils, ending with mature contractile myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent myofibrils from transitioning to mature myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature myofibrils.
脊椎动物横纹肌的肌原纤维从头组装经历三个明显的步骤，首先是在原肌小节中几种非肌肉和肌肉蛋白的最小肌节排列，然后是新生肌小节中额外蛋白的插入和组织增加，最后是成熟的收缩肌小节。在寻找肌原纤维组装过程的控制因素时，我们发现，新生肌小节向成熟肌小节的转变可以被泛素蛋白酶体系统 (UPS) 的三种不同功能的抑制剂所阻止。首先，抑制泛素途径到 E3 Cullin 连接酶，这些酶会使蛋白质泛素化，导致肌原纤维生成在新生肌小节阶段停止。其次，抑制 p97 蛋白对泛素化蛋白的提取也会导致肌原纤维生成在新生肌小节阶段停止。第三，蛋白酶体的蛋白水解作用抑制剂也阻止了新生肌小节向成熟肌小节的转变。相比之下，自噬或溶酶体的抑制剂并不影响肌原纤维的生成。为了探究 UPS 抑制剂在肌原纤维生成过程中的差异，我们通过光漂白后荧光恢复分析了两种选定的肌节蛋白（肌球蛋白 II 重链和轻链）的交换率。在 p97 和蛋白酶体抑制剂存在的情况下，这两种肌球蛋白蛋白的每一种在新生肌小节阶段的动力学都会降低，但在成熟肌小节阶段不受影响。从新生肌小节向成熟肌小节组装的转变中肌小节的稳定性增加表明，肌球蛋白 II 蛋白的动力学和选择性破坏对肌小节的重塑很重要。
