Genetic dissection of the phospholipid hydroperoxidase activity of yeast Gpx3 reveals its functional importance
Genetic dissection of the phospholipid hydroperoxidase activity of yeast Gpx3 reveals its functional importance
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DOI：
10.1074/jbc.m408340200
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发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Avery, SV
中科院分区：
文献类型：
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作者：
Avery, AM;Willetts, SA;Avery, SV
酿酒酵母表达多种磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）样蛋白的经典谷胱甘肽过氧化物酶（cGPx）的情况下，提供了一个独特的系统解剖这些酶在体内的作用。Gpx 3（Orp 1/PHGpx 3）蛋白将氢过氧化物信号转导至转录因子Yap 1，这一功能可以解释大多数GPX依赖性表型。为了验证这一假设，并确定哪些功能的Gpx 3可以共享cGPx样酶，我们构建了一种新的cGPx样酵母酶，cGpx 3。我们证实，cGPx之间保守的“间隙”序列，但没有对齐的PHGPx序列是这些酶的结构和功能差异的主要原因。对cGPx底物的过氧化物酶活性是高的cGpx 3的构建体，这是多聚体，并有一个过氧化物酶催化机制不同于Gpx 3，但cGpx 3是有缺陷的磷脂氢过氧化物酶和信号传导活动。cGpx 3没有补充的gpxDelta突变体的脂质过氧化的敏感性，并赋予Gpx 3的脂质过氧化的抗性是独立的Yap 1，建立了Gpx 3磷脂过氧化氢酶活性的功能作用。使用cGpx 3和Gpx 3与其他结构结合探测脂质过氧化作为一种毒性机制之间的比较，我们还确定，脂质过氧化依赖的过程是细胞镉毒性的主要原因。结果表明，磷脂过氧化氢酶和Yap 1介导的Gpx 3的信号传导活动具有独立的功能作用，虽然这两种功能依赖于cGPx样亚基相互作用位点的情况下，结果更清楚地解决了GPx样酶的差异选择性进化的潜在驱动因素。
Saccharomyces cerevisiae expresses multiple phospholipid hydroperoxide glutathione peroxidase (PHGPx)-like proteins in the absence of a classical glutathione peroxidase (cGPx), providing a unique system for dissecting the roles of these enzymes in vivo. The Gpx3 (Orp1/PHGpx3) protein transduces the hydroperoxide signal to the transcription factor Yap1, a function that could account for most GPX-dependent phenotypes. To test this hypothesis and ascertain what functions of Gpx3 can be shared by cGPx-like enzymes, we constructed a novel cGPx-like yeast enzyme, cGpx3. We confirmed that the "gap" sequences conserved among cGPxs but absent from aligned PHGPx sequences are the principal cause of the structural and functional differences of these enzymes. Peroxidase activity against a cGPx substrate was high in the cGpx3 construct, which was multimeric and had a peroxidase catalytic mechanism distinct from Gpx3; but cGpx3 was defective for phospholipid hydroperoxidase and signaling activities. cGpx3 did not complement the sensitivity to lipid peroxidation of a gpxDelta mutant, and the resistance to lipid peroxidation conferred by Gpx3 was independent of Yap1, establishing a functional role for Gpx3 phospholipid hydroperoxidase activity. Using the comparison between cGpx3 and Gpx3 in conjunction with other constructs to probe lipid peroxidation as a toxicity mechanism, we also ascertained that lipid peroxidation-dependent processes are a principal cause of cellular cadmium toxicity. The results demonstrate that phospholipid hydroperoxidase and Yap1-mediated signaling activities of Gpx3 have independent functional roles, although both functions depend on the absence of cGPx-like subunit interaction sites, and the results resolve more clearly the potential drivers of the differential selective evolution of GPx-like enzymes.