Hussain Sabah N A, Matar Ghassan, Barreiro Esther, Florian Maria, Divangahi Maziar, Vassilakopoulos Theodoros
Critical Care and Respiratory Divisions, Royal Victoria Hospital, McGill University Health Centre, McGill University, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada.
Am J Physiol Lung Cell Mol Physiol. 2006 May;290(5):L996-1003. doi: 10.1152/ajplung.00337.2005.
Although 4-hydroxy-2-nonenal (HNE, a product of lipid peroxidation) is a major cause of oxidative damage inside skeletal muscles, the exact proteins modified by HNE are unknown. We used two-dimensional electrophoresis, immunoblotting, and mass spectrometry to identify selective proteins targeted by HNE inside the diaphragm of rats under two conditions: severe sepsis [induced by E. coli lipopolysaccharides (LPS)] and during strenuous muscle contractions elicited by severe inspiratory resistive loading (IRL). Diaphragm HNE-protein adduct formation (detected with a polyclonal antibody) increased significantly after 1 and 3 h of LPS injection with a return to baseline values thereafter. Similarly, HNE-protein adduct formation inside the diaphragm rose significantly after 6 but not 3 h of IRL. Mass spectrometry analysis of HNE-modified proteins revealed enolase 3b, aldolase and triosephosphate isomerase 1, creatine kinase, carbonic anyhdrase III, aconitase 2, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein-beta. Measurements of in vitro enolase activity in the presence of pure HNE revealed that HNE significantly attenuated enolase activity in a dose-dependent fashion, suggesting that HNE-derived modifications have inhibitory effects on enzyme activity. We conclude that lipid peroxidation products may inhibit muscle contractile performance through selective targeting of enzymes involved in glycolysis, energy production as well as CO(2) hydration.
尽管4-羟基-2-壬烯醛（HNE，脂质过氧化的产物）是骨骼肌内氧化损伤的主要原因，但被HNE修饰的确切蛋白质尚不清楚。我们使用二维电泳、免疫印迹和质谱法，在两种情况下鉴定大鼠膈肌中被HNE靶向的选择性蛋白质：严重脓毒症[由大肠杆菌脂多糖（LPS）诱导]和严重吸气性阻力负荷（IRL）引起的剧烈肌肉收缩期间。注射LPS 1小时和3小时后，膈肌中HNE-蛋白质加合物的形成（用多克隆抗体检测）显著增加，此后恢复到基线值。同样，IRL 6小时后膈肌内HNE-蛋白质加合物的形成显著增加，但3小时后未增加。对HNE修饰蛋白质的质谱分析显示有烯醇化酶3b、醛缩酶、磷酸丙糖异构酶1、肌酸激酶、碳酸酐酶III、乌头酸酶2、二氢硫辛酰胺脱氢酶和电子传递黄素蛋白-β。在纯HNE存在下体外测量烯醇化酶活性表明，HNE以剂量依赖方式显著减弱烯醇化酶活性，这表明HNE衍生的修饰对酶活性有抑制作用。我们得出结论，脂质过氧化产物可能通过选择性靶向参与糖酵解、能量产生以及CO₂水合作用的酶来抑制肌肉收缩性能。
