Acquired mtDNA depletion and nucleoside reverse transciptase inhibitors

描述（由申请人提供）：该项目定义了由核苷逆转录酶抑制剂（NRTI）引起的HIV/AIDS获得性线粒体（mt）DNA耗竭的体内机制。由于嘧啶NRTI如齐多夫定（AZT）和司他夫定（d4 T），HIV/AIDS的生存率得到改善，但NRTI线粒体毒性限制了治疗，并表现为mtDNA耗竭和器官功能障碍。“DNA pol gamma假说”强调了在获得性mtDNA耗竭的机制中NRTI磷酸化（至活性部分）和NRTI三磷酸（-TP）对DNA pol gamma（mtDNA复制酶）的抑制。细胞激酶控制线粒体核苷酸和NRTI磷酸化。嘧啶NRTI与dThd竞争通过胸苷激酶（细胞质TK 1和线粒体TK 2亚型）磷酸化为单磷酸（MP），并通过胸苷酸激酶（TMPK）磷酸化为二磷酸（DP）。工作假设指出：野生型（WT）TK 2或WT TMPK的转基因（TG）过表达通过增加线粒体dTTP质量（与酶丰度增加平行）促进mtDNA复制。具有TMPK突变体（“功能获得”）的TG对dTMP磷酸化有活性，但对AZTMP表现出增强的活性。在不存在AZT的情况下，这些TG各自增加dTTP丰度。AZT治疗后，AZT-DP丰度显著增加，并被输入线粒体磷酸化为AZT-TP。相反，携带特异性TK 2点突变的TG降低TK 2活性（“功能丧失”）。这些后一种TG通过限制线粒体dTMP用于下游线粒体内加工来消耗mtDNA。AZT处理通过与dThd竞争TK 2的磷酸化而增强mtDNA的耗竭。线粒体AZT-TP与dTTP竞争通过DNA pol γ掺入mtDNA。这会抑制mtDNA复制，导致mtDNA缺失和突变，线粒体功能障碍和器官功能障碍。AIM 1定义了使用具有心脏靶向、条件性、WT TMPK、嵌合TMPK和突变TMPK过表达的TG从NRTI（如AZT）中去除mtDNA的分子、病理和生理事件。AIM 2定义了使用具有心脏靶向、条件性、WT TK 2和突变TK 2过表达的TG从NRTI（如AZT）中清除mtDNA的分子、病理和生理事件。实验为改善艾滋病抗逆转录病毒药物治疗提供了新的见解。

DESCRIPTION (provided by applicant): This project defines in vivo mechanisms of acquired mitochondrial (mt) DNA depletion caused by nucleoside reverse transcriptase inhibitors (NRTI) for HIV/AIDS. Survival with HIV/AIDS improved because of pyrimidine NRTIs like zidovudine (AZT) and stavudine (d4T), but NRTI mitochondrial toxicity limits therapy and manifests as mtDNA depletion and organ dysfunction. The "DNA pol gamma hypothesis" underscores NRTI phosphorylation (to active moieties) and inhibition of DNA pol gamma (the mtDNA replicase) by NRTI triphosphates (-TP) in the mechanism of acquired mtDNA depletion. Cellular kinases control mitochondrial nucleotide and NRTI phosphorylations. Pyrimidine NRTIs compete with dThd for phosphorylation to monophosphates (MP) by thymidine kinase (cytoplasmic TK1 and mitochondrial TK2 isoforms) and to diphosphates (DP) by thymidylate kinase (TMPK). The working hypothesis states: Transgenic (TG) over-expression of wild type (WT) TK2 or WT TMPK promotes mtDNA replication by increasing the mitochondrial dTTP mass in parallel with increased enzyme abundance. TGs with TMPK mutants ("gain of function") are active with dTMP phosphorylation, but exhibit enhanced activity with AZTMP. In absence of AZT, these TGs each increases dTTP abundance. With AZT therapy, AZT-DP abundance increases dramatically and is imported into mitochondria for phosphorylation to AZT-TP. Conversely, TGs harboring specific TK2 point mutations decrease TK2 activity ("loss of function"). These latter TGs deplete mtDNA by limiting mitochondrial dTMP for down- stream intra-mitochondrial processing. AZT treatment here enhances mtDNA depletion by AZT's competition with dThd for phosphorylation by TK2. Mitochondrial AZT-TP competes with dTTP for incorporation into mtDNA by DNA pol gamma. This inhibits mtDNA replication, causes mtDNA depletion and mutation, mitochondrial dysfunction, and organ dysfunction. AIM 1 defines molecular, pathological and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpression of WT TMPK, chimeric TMPK, and mutant TMPK. AIM 2 defines molecular, pathological, and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpression of WT TK2 and mutant TK2. Experiments offer insights into improving therapy with antiretrovirals used in AIDS.