Department of Neurology, University of Crete, School of Health Sciences, Section of Medicine, Voutes, 71003 Heraklion, Crete, Greece.
Neurochem Int. 2012 Sep;61(4):463-9. doi: 10.1016/j.neuint.2012.06.006. Epub 2012 Jun 16.
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.
人类谷氨酸脱氢酶 (hGDH) 存在两种高度同源的同工酶，具有明显不同的调节和组织表达谱：一种是管家 hGDH1 同工酶，由 GLUD1 基因编码；另一种是 hGDH2 同工酶，由 GLUD2 基因编码。有证据表明，这两种同工酶都是作为包含 53 个氨基酸长的 N 端前导肽的前酶合成的，该前导肽在易位到线粒体时被切割。然而，这种 GDH 信号肽明显大于大多数核 DNA 编码的线粒体蛋白的前导序列，其前导序列通常包含 17-35 个氨基酸，并且它们通常形成单个两性 α-螺旋。为了解码 hGDH 线粒体靶向所必需的结构元件，我们对其前导序列进行了二级结构分析。这些分析以 82%的准确率预测，两个前导肽均带正电荷，并且形成两个至三个 α-螺旋，中间有环隔开。hGDH2 的第一个 α-螺旋具有很强的两亲性，既带有正电荷表面，又带有疏水面。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）。用 pDsRed2-Mito 线粒体靶向载体共转染后，激光共聚焦扫描显微镜显示，前导序列的完全缺失阻止了酶进入线粒体，导致其滞留在细胞质中。仅缺失第一个强两亲性的 α-螺旋也足以阻止截短蛋白的线粒体定位。此外，保留第二个和/或第三个假定 α-螺旋的截短前导序列未能恢复 hGDH2 的线粒体导入。因此，第一个 N 端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 GDH 大线粒体靶向信号的进化机制具有启示意义。
