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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import
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Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

机译：人GLUD2谷氨酸脱氢酶前导序列中的α螺旋结构负责线粒体的导入。

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摘要

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.
机译：人谷氨酸脱氢酶（hGDH）存在两种高度同源的同工型，具有不同的调节和组织表达特征：由GLUD1基因编码的管家hGDH1同蛋白和由GLUD2基因编码的hGDH2同工酶。有证据表明，两种同工酶都合成为含有53个氨基酸长的N末端前导肽的原酶，该肽经易位进入线粒体后被切割。但是，该GDH信号肽比大多数核DNA编码的线粒体蛋白要大得多，后者的前导序列通常包含17-35个氨基酸，并且它们通常形成单个两亲性α-螺旋。为了解码对人类GDH的线粒体靶向至关重要的结构元件，我们对其前导序列进行了二级结构分析。这些分析以82％的准确度预测，两个前导肽均带正电，并且形成两个至三个由中间环隔开的α螺旋。 hGDH2的第一个α螺旋是强两亲性的，既显示带正电的表面又显示疏水的平面。然后，我们构建了GLUD2-EGFP缺失突变体，并将其用于转染三种哺乳动物细胞系（HEK293，COS 7和SHSY-5Y）。与pDsRed2-Mito线粒体靶向载体共转染后，共聚焦激光扫描显微镜显示，整个前导序列的缺失阻止了酶进入线粒体，从而使其保留在细胞质中。仅删除第一个强两亲性α-螺旋也足以防止截短的蛋白质的线粒体定位。此外，保留第二和/或第三推定的α-螺旋的截短的前导序列未能恢复hGDH2的线粒体导入。因此，第一个N末端的α螺旋结构对于hGDH2的线粒体导入至关重要，这些发现可能对理解导致人类GDHs的大线粒体靶向信号的进化机制具有重要意义。

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