Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely on a means for efficient and long-lasting circulation of the TP enzyme. In this study we exploit lentiviral transduction of hematopoietic stem cells and an erythroid cell line (BEL-A) to generate reticulocytes that contain active TP. Significant loss of overexpressed TP during erythroid differentiation can be reduced by addition of the ubiquitination inhibitor MG132. However, the ubiquitination sites are located in the substrate binding site in human TP, and their removal abolished enzyme activity. Examination of the TP structure and mechanism suggested that these sites are only exposed in the absence of substrate. We show that supplementation of culture media with thymidine during differentiation reduces enzyme degradation, doubling the amount of TP retained in reticulocytes. This study provides proof of principle that therapeutic reticulocytes expressing TP can be generated in vitro and that ubiquitin-mediated degradation can be subverted through masking ubiquitination sites to ensure retention of human TP in reticulocytes following erythroid differentiation.

中文翻译：

---

胸苷磷酸化酶在网织红细胞中的表达和保留，作为MNGIE的新型治疗方法。

线粒体神经胃肠道脑病（MNGIE）是由胸苷磷酸化酶（TP）缺乏引起的一种罕见的常染色体代谢性疾病。对这种疾病的成功的治疗性干预依赖于一种有效而持久的TP酶循环的手段。在这项研究中，我们利用造血干细胞和红系细胞系（BEL-A）的慢病毒转导来产生含有活性TP的网状细胞。通过添加泛素化抑制剂MG132，可以减少红系分化过程中过表达TP的明显损失。但是，泛素化位点位于人TP的底物结合位点，其去除消除了酶的活性。TP结构和机理的研究表明，这些位点仅在没有底物的情况下才暴露。我们显示，在分化过程中用胸苷补充培养基可降低酶降解，使网织红细胞中保留的TP量增加一倍。这项研究提供了原理上的证据，即可以在体外产生表达TP的治疗性网织红细胞，并且可以通过掩盖泛素化位点来破坏泛素介导的降解，以确保人类TP在红系分化后保留在网织红细胞中。