A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta
A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta
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DOI：
10.1016/s1525-0016(03)00130-8
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发表时间：
2003-06-01
影响因子：
12.4
通讯作者：
Payne, RM
中科院分区：
文献类型：
--
作者：
Del Gaizo, V;Payne, RM
核和线粒体基因组的突变可能导致线粒体功能缺陷。迄今为止，无论是病毒载体还是非病毒载体，都很难通过外源蛋白质或基因转移来修复这些缺陷。我们假设 TAT 融合蛋白会穿过线粒体膜，并且将线粒体信号序列掺入 TAT 融合蛋白中将允许外源蛋白在线粒体中进行加工和定位。构建了 TAT-线粒体苹果酸脱氢酶信号序列（mMDH）增强绿色荧光蛋白（eGFP）融合蛋白。 TAT-mMDH-eGFP 允许将融合蛋白快速转导并定位到多种细胞类型的线粒体中。相比之下，没有线粒体信号序列的TAT-GFP可以快速转导到细胞和线粒体中，表现出伪一级动力学，但不会停留在那里。 5 天前注射 TAT-mMDH-eGFP 的小鼠在多种组织类型中可检测到 eGFP 活性。从肝脏中分离出的细胞质和线粒体部分的蛋白质印迹证实了 eGFP 定位于线粒体，并且 mMDH 转运肽被识别和加工。此外，注射到怀孕小鼠体内的 TAT-mMDH-eGFP 融合蛋白可以穿过胎盘，并且在胎儿和新生幼崽中均可检测到。含有线粒体信号序列的 TAT 融合蛋白是将蛋白质定位到线粒体的可行方法。
Mutations in nuclear and mitochondrial genomes can lead to defects in mitochondrial function. To date, repair of these defects with exogenous proteins or gene transfer has been difficult with either viral or nonviral vectors. We hypothesized that TAT fusion proteins would cross both mitochondrial membranes and that incorporation of a mitochondrial signal sequence into a TAT fusion protein would allow processing and localization of exogenous proteins in mitochondria. A TAT-mitochondrial malate dehydrogenase signal sequence (mMDH)-enhanced green fluorescent protein (eGFP) fusion protein was constructed. TAT-mMDH-eGFP allowed rapid transduction and localization of fusion protein into mitochondria of multiple cell types. In contrast, TAT-GFP, without a mitochondrial signal sequence, rapidly transduced into cells and mitochondria, displayed pseudo-first-order kinetics, but did not remain there. Mice injected 5 days prior with TAT-mMDH-eGFP had detectable eGFP activity in multiple tissue types. Western blotting of cytosolic and mitochondrial fractions isolated from their livers confirmed eGFP localization to mitochondria and that the mMDH transit peptide was recognized and processed. Furthermore, TAT-mMDH-eGFP fusion protein injected into pregnant mice crossed the placenta and was detectable in both the fetus and the newborn pups. TAT fusion proteins containing a mitochondrial signal sequence are a viable method to localize proteins to mitochondria.