In Vitro and in Silico Characterization of Peroxiredoxin 6 Modified by 4-Hydroxynonenal and 4-Oxononenal
In Vitro and in Silico Characterization of Peroxiredoxin 6 Modified by 4-Hydroxynonenal and 4-Oxononenal
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DOI：
10.1021/tx800244u
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发表时间：
2008-12-01
影响因子：
4.1
通讯作者：
Petersen, Dennis R.
中科院分区：
文献类型：
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作者：
Roede, James R.;Carbone, David L.;Petersen, Dennis R.
过氧化物氧还蛋白6（Peroxiredoxin 6，PRX6）属于1-Cys类过氧化物氧还蛋白，被认为是心肌、皮肤和肺等组织中重要的抗氧化蛋白。初步的体内蛋白质组学数据显示，PRX6是加合的4-羟基壬烯醛（4HNE）在大鼠的肝脏长期喂养含乙醇的饮食。本研究的目的是评估醛加合对PRX6过氧化物酶活性的体外影响，使用质谱法确定醛修饰的特定位点，并使用分子建模预测由于加合引起的构象变化。发现PRX6通过醛修饰对失活具有抗性;然而，加合蛋白的蛋白质印迹显示，4HNE和4-氧代壬烯醛（4ONE）均引起广泛的交联，导致高分子量物质。串联质谱法（ESI-LC-MS/MS）分析证明了多个修饰位点，但未观察到活性位点Cys（47）的加合。分子建模模拟表明，Cys（91）处的加合导致蛋白活性位点构象的变化，这可能限制4-HNE接近Cys（47）。Cys（91）-Lys（209）交联加合物可以通过限制亲电试剂的进入或阻止催化三联体内的重要氨基酸相互作用来提供使蛋白质变性所需的构象变化。
Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys(47) was not observed. Molecular modeling simulations indicated that adduction at Cys(91) results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys(47). The Cys(91)-Lys(209) cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.