Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida, United States of America.
PLoS Genet. 2022 Dec 27;18(12):e1010574. doi: 10.1371/journal.pgen.1010574. eCollection 2022 Dec.
Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3β, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3β co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.
骨骼肌中的许多分子和生理过程会根据日常活动/休息周期发生昼夜节律依赖性波动。这些周期性过程背后的昼夜调节机制，特别是在转录后水平上，尚未得到很好的定义。此前，我们报道了昼夜节律 E3 连接酶 FBXL21 与 GSK-3β 一起介导肌节蛋白 TCAP 的节律性降解，并且携带 Fbxl21 功能降低等位基因的 Psttm 小鼠表现出纤维直径减小和肌肉功能受损。为了进一步阐明 FBXL21 在骨骼肌中的调节功能，我们研究了另一种肌节蛋白 Myozenin1（MYOZ1），我们从酵母 2 杂交筛选中鉴定出它是 FBXL21 的结合蛋白。我们表明，FBXL21 与 MYOZ1 的结合导致泛素化介导的蛋白酶体降解。发现 GSK-3β 的共表达和抑制分别加速和减缓 FBXL21 介导的 MYOZ1 降解。此前，MYOZ1 已被证明抑制钙调神经磷酸酶/NFAT 信号通路，该信号通路对肌肉分化很重要。因此，与对照 C2C12 细胞相比，Fbxl21 KO 和 MyoZ1 KO 的 C2C12 细胞的肌生成分化分别受损和增强，同时对 NFAT 核定位和 NFAT 靶基因表达产生不同的影响。重要的是，在 Psttm 小鼠中，与野生型小鼠相比，NFAT2 核定位的水平和昼夜节律均显著降低，与肌肉分化相关的 NFAT 靶基因的昼夜表达也明显减弱。此外，Psttm 小鼠表现出肌节结构的明显破坏，Z 线中 MYOZ1 的积累明显过多。总之，我们的研究表明 FBXL21 通过调节 MYOZ1 降解和 NFAT2 信号在肌节结构和肌肉分化中起着关键作用。
