摘要
Skeletal muscle atrophy is defined as a decrease in muscle size and occurs due to disparate physiological conditions, including aging, immobilization, and corticosteroid exposure. TSSK6 activating co-chaperone (TSACC) was identified as a gene that is significantly upregulated in skeletal muscle in response to denervation. To confirm Tsacc expression in skeletal muscle, the Tsacc cDNA was cloned from cultured myoblast cells leading to the discovery of a novel alternative splice-variant that is 49 amino acids shorter than the known full-length transcript. Quantitative PCR (qPCR) confirmed that Tsacc is expressed in skeletal muscle with expression increasing as muscle cells undergo differentiation. Characterization of Tsacc transcriptional activity revealed that Tsacc gene activity is modulated by myogenic regulatory factors (MRFs) and is significantly induced by MyoD. Moreover, sub-cellular localization was assessed by confocal microscopy, which showed that the two Tsacc proteins produced by the two alternatively spliced transcripts had distinct localization patterns. Tsacc-FL localized exclusively to the cytoplasm of muscle cells while Tsacc-novel localized diffusely within the cell, appearing in both the nucleus and cytoplasm of muscle cells. Finally, Western blot analysis revealed that ectopic expression of the full-length Tsacc transcript in C2C12 resulted in significant inhibition of conical markers of muscle differentiation, including MyHC and myogenin while the novel Tsacc transcript did not. The impact of Tsacc on the AKT signaling pathway, which is known to participate in the regulation of muscle size, was also assessed. Overexpression of either the full-length or novel Tsacc transcript was found to modulate the AKT signaling pathway in cultured muscle cells. Understanding the role of Tsacc will further our understanding of the molecular and genetic mechanisms of the muscle wasting and potentially provide new therapeutic targets for mitigating the negative outcomes associated with muscle atrophy.
摘要译文
骨骼肌萎缩定义为肌肉大小的降低，并且由于不同的生理状况（包括衰老，固定和皮质类固醇暴露）而发生。TSSK6激活共酮（TSACC）被鉴定为一种基因，该基因在骨骼肌中显着上调，以响应于神经膜。为了确认骨骼肌中的TSACC表达，从培养的肌细胞细胞中克隆TSACC cDNA，从而发现了一种新型的替代剪接变量，该替代剪接变体比已知的全长转录物短49个氨基酸。定量PCR（QPCR）证实TSACC在骨骼肌中表达，表达随着肌肉细胞的经历而增加。TSACC转录活性的表征表明，TSACC基因活性是由肌源性调节因子（MRF）调节的，并由MYOD显着诱导。此外，通过共聚焦显微镜评估了亚细胞定位，这表明由两个剪接的转录本产生的两种TSACC蛋白具有不同的定位模式。TSACC-FL仅位于肌肉细胞的细胞质中，而TSACC-Novel局部扩散在细胞内，出现在肌肉细胞的细胞核和细胞质中。最后，Western印迹分析表明，C 2 C 12中全长TSACC转录本的异位表达导致对肌肉分化的锥形标记的显着抑制，包括MyHC和肌蛋白，而新型TSACC转录本没有。还评估了TSACC对参与肌肉大小调节的AKT信号通路的影响。发现全长或新型TSACC转录本的过表达可调节培养的肌肉细胞中的Akt信号通路。了解TSACC的作用将进一步理解肌肉浪费的分子和遗传机制，并可能提供新的治疗靶标，以减轻与肌肉萎缩相关的负面结果。
Tello, Tala Shourbagi. Identification and Characterization of the TSSK6 Activating Co-Chaperone (TSACC) Gene In Skeletal Muscle[D]. US: University of North Florida, 2022