Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers
Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers
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DOI：
10.1152/ajpcell.00180.2012
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发表时间：
2012-09-01
影响因子：
5.5
通讯作者：
Lamb, Graham D.
中科院分区：
文献类型：
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作者：
Larkins, Noni T.;Murphy, Robyn M.;Lamb, Graham D.
Larkins NT，Murphy RM，Lamb GD。温度、氧化应激和磷酸化对骨骼肌纤维中热休克蛋白结合的影响。美国生理学杂志细胞生理学303：C654-C665，2012年。首次发表于2012年7月3日; doi：10.1152/ajpcell.00180.2012。热休克蛋白（HSPs）有助于在应激情况下维持细胞功能，但控制其与靶蛋白相互作用的过程尚未明确。本研究检测了各种应激后骨骼肌纤维中HSP 72、HSP 25和α B-晶体蛋白的结合。大鼠比目鱼肌（SOL）和趾长伸肌（EDL）在离体条件下接受热应激或强疲劳刺激。通过显微切割将浅表纤维“剥皮”，并从暴露于生理细胞内溶液10- 30分钟后的洗脱程度评估HSP扩散性。在来自非应激（对照）SOL肌肉的纤维中，>80%的每种HSP容易扩散。然而，在将肌肉加热至40 ℃持续30分钟后，类似于95%的HSP 25和α B-晶状体蛋白在非膜肌原纤维位点紧密结合，而HSP 72仅在加热处理至≥ 44 ℃后在膜位点结合。还原型与氧化型细胞质谷胱甘肽（GSH：GSSG）的比例在加热肌肉至40度和45度C后分别下降了约2倍和4倍。还原剂二硫苏糖醇逆转热休克蛋白72结合在加热的肌肉，但对其他热休克蛋白没有影响。SOL肌肉在体外的强烈刺激，足以引起大量的氧化相关的损失的最大力量和GSH：GSSG的比例下降约四倍，没有影响的任何HSP的扩散性。当皮肤纤维从热处理的肌肉沐浴额外的外源性HSP 72，总结合增加约2和10倍，分别在SOL和EDL纤维，可能反映了相对肌（内）浆网Ca 2 +-ATP酶泵密度在两种纤维类型。在Ser 59的α B-晶体蛋白和Ser 85的HSP 25的磷酸化增加与热处理，但似乎没有确定HSP结合。这些发现突出了控制HSP 72和两种小HSPs结合的过程中的主要差异。结合不直接相关的细胞质氧化状态，但半胱氨酸残基的氧化影响HSP 72结合。
Larkins NT, Murphy RM, Lamb GD. Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers. Am J Physiol Cell Physiol 303: C654-C665, 2012. First published July 3, 2012; doi:10.1152/ajpcell.00180.2012.-Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and alpha B-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40 degrees C for 30 min similar to 95% of HSP25 and alpha B-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to >= 44 degrees C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40 degrees and 45 degrees C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo) plasmic reticulum Ca2+-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on alpha B-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.