Gaffney Christopher J, Shephard Freya, Chu Jeff, Baillie David L, Rose Ann, Constantin-Teodosiu Dumitru, Greenhaff Paul L, Szewczyk Nathaniel J
MRC/ARUK Centre for Musculoskeletal Ageing Research, Faculty of Medicine and Health Sciences University of Nottingham Nottingham NG7 2UH UK.
Department of Molecular Biology and Biochemistry Simon Fraser University Burnaby BCV5A 1S6 Canada; Department of Medical Genetics University of British Columbia Vancouver BCV6T 1Z4 Canada.
J Cachexia Sarcopenia Muscle. 2016 May;7(2):181-92. doi: 10.1002/jcsm.12040. Epub 2015 Jun 4.
Declines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC-105/degenerin channel activation produced muscle protein degradation via an unknown mechanism.
Generation of transgenic and double mutant C. elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function.
unc-105 gain-of-function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let-2 mutants that gate the hyperactive UNC-105 channel. Degradation is not suppressed by interventions suppressing proteasome-mediated, autophagy-mediated, or calpain-mediated degradation nor by suppressors of degenerin-induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced-3 or ced-4. Adult unc-105 muscles display a time-dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED-4, which is sufficient to activate CED-3 in vitro, are observed in unc-105 mitochondrial isolations.
Constitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia.
在各种临床人群中均发现骨骼肌结构和功能下降，但对于这些个体中导致肌肉衰退的肌内蛋白水解途径仍了解相对较少。线虫秀丽隐杆线虫已被开发成为一种用于识别和理解这些途径的模型。最近，有报道称UNC-105/退化素通道激活通过未知机制导致肌肉蛋白质降解。
利用转基因和双突变秀丽隐杆线虫的生成、RNA干扰和药物处理来评估控制蛋白质降解的分子事件。蛋白质印迹法用于测量蛋白质含量。利用阳离子染料和三磷酸腺苷（ATP）生成测定法来测量线粒体功能。
unc-105功能获得性突变体表现出异常的肌肉蛋白质降解和运动缺陷；在基因内回复突变体和控制过度活跃的UNC-105通道的let-2突变体中，这两种情况均有所减轻。抑制蛋白酶体介导的、自噬介导的或钙蛋白酶介导的降解的干预措施，以及退化素诱导的神经退行性变的抑制剂，均不能抑制降解。用半胱天冬酶抑制剂或针对ced-3或ced-4的RNA干扰处理可减少蛋白质降解，但不能减少运动缺陷。成年unc-105肌肉显示出线粒体网状结构的时间依赖性碎片化，这与线粒体膜电位受损相关，并且与最大ATP生成速率降低相关。在unc-105线粒体分离物中观察到CED-4水平降低，而CED-4在体外足以激活CED-3。
持续的阳离子流入肌肉似乎由于线粒体功能障碍而导致胞质蛋白的半胱天冬酶降解，这可能与衰老和肌肉减少症有关。
