Zabit Samar, Melloul Orly, Lichtenstein Michal, Seifert Erin L, Lorberboum-Galski Haya
Department of Biochemistry and Molecular Biology, The Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel.
MitoCare Center, Department of Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Int J Mol Sci. 2025 May 5;26(9):4379. doi: 10.3390/ijms26094379.
The mitochondrial phosphate carrier (mPiC), encoded by the nuclear gene , is synthesized with an N-terminus mitochondrial targeting sequence (MTS), enabling its import into the mitochondria. mPiC imports inorganic phosphate (P) into the mitochondrial matrix for ATP production and other matrix phosphorylation reactions, as well as regulates mitochondrial Ca uptake and buffering of matrix Ca. PiC also imports copper (Cu), crucial to COX subunit holoenzyme assembly. Variants in exist and lead to mPiC deficiency (MPCD), cause a rare autosomal recessive disease with no current cure; patients with MPCD usually die within the first year of life. We have developed a novel therapeutic approach using TAT-mPiC fusion protein for cellular delivery since the TAT peptide enables delivery of proteins across biological membranes. We designed, produced, and purified the TAT-mPiC fusion protein. The fusion protein is delivered into the mitochondria and localizes within the mIM, its natural cellular location, as a processed protein. Treatment of mPiC-knockdown cells with TAT-mPiC fusion protein increased cell growth and improved bioenergetic capabilities, as measured by oxygen consumption rate (OCR), ATP production, and reduction in lactate secretion. Most importantly, TAT-mPiC restored P and Cu delivery into the mitochondrial matrix. TAT-mPiC fusion protein also restored the mitochondrial activity of cells harboring various mitochondrial defects. This study presents the first successful delivery of a mitochondrial transmembrane carrier using the TAT-fusion system, offering a potential early treatment strategy for newborns with mPiC deficiency.
线粒体磷酸盐载体（mPiC）由核基因编码，合成时带有N端线粒体靶向序列（MTS），使其能够导入线粒体。mPiC将无机磷酸盐（P）导入线粒体基质用于ATP生成及其他基质磷酸化反应，还能调节线粒体对钙的摄取和基质钙的缓冲。PiC也能导入对细胞色素c氧化酶（COX）亚基全酶组装至关重要的铜（Cu）。存在相关变体并导致mPiC缺乏症（MPCD），这是一种罕见的常染色体隐性疾病，目前无法治愈；MPCD患者通常在出生后第一年内死亡。由于TAT肽能够使蛋白质穿过生物膜，我们开发了一种使用TAT - mPiC融合蛋白进行细胞递送的新型治疗方法。我们设计、生产并纯化了TAT - mPiC融合蛋白。该融合蛋白作为一种加工后的蛋白质被递送到线粒体中，并定位在内膜间隙（mIM），即其天然的细胞位置。用TAT - mPiC融合蛋白处理mPiC基因敲低的细胞可增加细胞生长，并改善生物能量能力，这通过耗氧率（OCR）、ATP生成以及乳酸分泌减少来衡量。最重要的是，TAT - mPiC恢复了P和Cu向线粒体基质的递送。TAT - mPiC融合蛋白还恢复了具有各种线粒体缺陷的细胞的线粒体活性。本研究首次成功利用TAT融合系统递送线粒体跨膜载体，为患有mPiC缺乏症的新生儿提供了一种潜在的早期治疗策略。
