Alpha-actinins in normal and diseased muscle

描述（由申请人提供）：α -肌动蛋白是肌动蛋白结合蛋白，包括组织肌动蛋白丝的细胞骨架异构体和代表肌肉z线主要结构成分的肌肉异构体异构体。基于它们与许多与z线和细丝相关的结构和信号蛋白的相互作用，肌聚性α -肌动蛋白可能在骨骼肌中执行静态功能，以维持有序的肌纤维阵列，以及协调肌纤维结构和功能的可能调节功能。在人类中，有两个基因编码骨骼肌α -肌动素，ACTN2和ACTN3。-肌动蛋白-2存在于所有骨骼肌纤维中，而-肌动蛋白-3仅在快速（2型）纤维中表达。我们之前已经表明，由于ACTN3 （R577X）中一个过早停止密码子的纯合性，α -肌动蛋白-3缺乏症在一般人群中很常见。α -肌动蛋白-3的缺失与疾病表型无关，α -肌动蛋白-2很可能能够“补偿”人类α -肌动蛋白-3的缺失。然而，在同型异型功能上一定存在细微的差异，因为我们发现，与对照组相比，专业短跑运动员的577XX （α -肌动蛋白-3无效）基因型的频率极低（p<0.0001），而优秀耐力运动员的577XX的频率很高。我们现在已经证明，α -肌动蛋白-2的转录敲除，而α -肌动蛋白-3的转录敲除，会导致纤维型基因表达的变化，α -肌动蛋白-2的转录敲除，而α -肌动蛋白-3的转录敲除，对小鼠胚胎是致命的。我们在这里提出的主要工作假设是，各种α -肌动蛋白异构体在结构上进化出了微妙的差异，使它们能够执行截然不同的功能。本申请拟利用细胞培养和斑马鱼发育模型以及转基因和敲除小鼠的转录敲低和拯救实验，研究α -肌动蛋白-2和-3的差异功能及其对快肌和慢肌独特生理作用的贡献。本提案中描述的实验将对我们理解α -肌动蛋白异构体在正常骨骼肌生物学中的不同作用，以及这些异构体可能影响正常人类健康和表现变化的机制做出重大贡献。我们的结果也可能导致对α -肌动蛋白基因型在调节和/或引起人类神经肌肉疾病中可能发挥的作用的深入了解。

DESCRIPTION (provided by applicant): The alpha-actinins are actin-binding proteins that include both cytoskeletal isoforms that organize actin filaments, and sarcomeric isoforms that represent major structural components of muscle Z-lines. Based on their interactions with many structural and signaling proteins associated with the Z-line and thin filament, sarcomeric alpha-actinins likely perform a static function in skeletal muscle to maintain the ordered myofibrillar array, as well as possible regulatory functions in coordinating myofiber structure and function. In humans, two genes encode the skeletal muscle alpha-actinins, ACTN2 and ACTN3. alpha-Actinin-2 is present in all skeletal muscle fibers, whereas alpha-actinin-3 is expressed only in fast (type 2) fibers. We have previously shown that alpha- actinin-3 deficiency is common in the general population due to homozygosity for a premature stop codon in ACTN3 (R577X). Absence of alpha-actinin-3 is not associated with a disease phenotype and it is likely that alpha- actinin-2 is able to "compensate" for the absence of alpha-actinin-3 in humans. Nevertheless, there must be subtle differences in isoform function as we have found that specialist sprint athletes have an extremely low frequency of the 577XX (alpha-actinin-3 null) genotype compared to controls, (p<0.0001), whereas elite endurance athletes have a high frequency of 577XX. We have now shown that transcriptional knockdown of alpha-actinin -2, but not alpha-actinin -3, results in changes in fiber type gene expression, and that knockout of alpha-actinin-2, but not -3, is embryonic lethal in mice. The central working hypothesis that we propose to test here is that the various alpha-actinin isoforms have evolved subtle differences in structure that allow them to perform distinctly different functions. This application proposes to utilize transcriptional knockdown and rescue experiments in cell culture and zebrafish developmental models, as well as transgenic and knockout mice, to study the differential functions of alpha-actinin-2 and -3 and their contributions to the unique physiological roles that fast and slow muscles perform. The experiments described in this proposal will make a significant contribution to our understanding of the differential roles of alpha-actinin isoforms in normal skeletal muscle biology, and the mechanism(s) by which these may influence normal human variations in fitness and performance. Our results may also lead to insights into the role(s) that alpha-actinin genotypes may play in modulating and/or causing human neuromuscular disease.