Laboratory of Pharmacology, School of Pharmacy, Faculty of Health Sciences , Aristotle University of Thessaloniki , Thessaloniki 54124 , Macedonia , Greece.
Adult Thalassemia Unit , Hippokrateion General Hospital , Thessaloniki 54642 , Greece.
Mol Pharm. 2018 Dec 3;15(12):5665-5677. doi: 10.1021/acs.molpharmaceut.8b00857. Epub 2018 Nov 12.
Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. β-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult β-globin chain. The resulting imbalance of α-/β-globin chains of adult hemoglobin (αβ) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant β-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature β-globin chain. Notably, the produced human recombinant β-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-β-globin protein interacts with the endogenous native α-like globins to form hemoglobin αβ-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-β-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for β-thalassemias.
蛋白质替代疗法（PRT）已被应用于治疗以蛋白质缺乏为特征的严重单基因/代谢疾病。在细胞内蛋白质缺失的疾病中，由于蛋白质无法穿过细胞膜，PRT 不易实施。相反，已经应用了基因治疗，但仍然收效有限。β-地中海贫血是严重的先天性血红蛋白病，其特征是成人β-球蛋白链缺乏或减少。由此导致的成人血红蛋白（αβ）中α-/β-球蛋白链的不平衡导致未配对的α-球蛋白链沉淀，并最终导致红细胞生成受损。由于蛋白转导结构域（PTD）技术已成为一种很有前途的治疗方法，我们生产了与人 TAT 肽融合的人重组β-球蛋白链，并成功地将其转导到缺乏成熟β-球蛋白链的人原红细胞 K-562 细胞中。值得注意的是，在类似条件下，用作内部阴性对照的不含 TAT 肽的人重组β-球蛋白链未能被转导到 K-562 细胞中。通过 SDS-PAGE、Western blot、共免疫沉淀和 LC-MS/MS 分析的计算机模拟研究表明，转导的重组融合 TAT-β-球蛋白蛋白与内源性天然α-样球蛋白相互作用，形成血红蛋白αβ样四聚体的程度有限。我们的研究结果提供了证据表明，重组 TAT-β-球蛋白可传入原红细胞 K-562 细胞，并可被视为β-地中海贫血的一种潜在的蛋白质替代治疗方法。
