Del Gaizo Victoria, MacKenzie James A, Payne R Mark
Division of Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1081, USA.
Mol Genet Metab. 2003 Sep-Oct;80(1-2):170-80. doi: 10.1016/j.ymgme.2003.08.017.
Disorders of mitochondrial function cause significant human disease and suffering. To date, correction of these mitochondria defects has depended on biochemical approaches and has not been achieved via gene therapies. Using previously described fusion proteins containing the transactivator of transcription (TAT) region from the HIV virus and green fluorescent protein (GFP), with and without a mitochondrial targeting sequence (MTS) from mitochondrial malate dehydrogenase (mMDH), we have investigated transduction across mitochondrial membranes. Both TAT-GFP and TAT-mMDH-GFP fusion proteins are protected from externally added protease when incubated with isolated mitochondria. Furthermore, both TAT fusion proteins rapidly enter cultured cells and transduce into mitochondria. However, the MTS allows processing of the fusion protein and is necessary for persistence in mitochondria over time. Neither degradation of import receptors nor disruption of the mitochondrial membrane potential or pH gradient inhibits protein transduction of either fusion protein. Furthermore, when injected into pregnant mice, TAT-mMDH-GFP is detectable throughout fetal and neonatal pups. These results indicate that TAT fusion proteins are able to traverse mitochondrial membranes through mechanisms that do not involve the regular import pathway, and that the addition of a MTS allows persistence of the fusion protein within mitochondria. TAT-MTS fusion proteins may represent a viable option as potential mitochondrial protein therapies.
线粒体功能紊乱会导致严重的人类疾病和痛苦。迄今为止，纠正这些线粒体缺陷依赖于生化方法，尚未通过基因疗法实现。利用先前描述的含有来自HIV病毒的转录反式激活因子（TAT）区域和绿色荧光蛋白（GFP）的融合蛋白，有或没有来自线粒体苹果酸脱氢酶（mMDH）的线粒体靶向序列（MTS），我们研究了跨线粒体外膜的转导。当与分离的线粒体一起孵育时，TAT-GFP和TAT-mMDH-GFP融合蛋白均受到保护，免受外部添加的蛋白酶的作用。此外，两种TAT融合蛋白都能迅速进入培养细胞并转导至线粒体。然而，MTS允许融合蛋白进行加工，并且对于随着时间的推移在线粒体中持续存在是必需的。导入受体的降解、线粒体膜电位或pH梯度的破坏均不会抑制任何一种融合蛋白的蛋白质转导。此外，当注射到怀孕小鼠体内时，在整个胎儿和新生幼崽中都可检测到TAT-mMDH-GFP。这些结果表明，TAT融合蛋白能够通过不涉及常规导入途径的机制穿过线粒体外膜，并且添加MTS可使融合蛋白在线粒体内持续存在。TAT-MTS融合蛋白可能是一种可行的潜在线粒体蛋白治疗选择。
