Acquired mtDNA depletion and nucleoside reverse transciptase inhibitors

描述（由申请人提供）：本项目定义由核苷类逆转录酶抑制剂（NRTI）引起的HIV/AIDS获得性线粒体（mt） DNA缺失的体内机制。由于嘧啶类NRTI如齐多夫定（AZT）和司他夫定（d4T），艾滋病毒/艾滋病患者的生存率提高，但NRTI线粒体毒性限制了治疗，表现为mtDNA耗竭和器官功能障碍。“DNA pol γ假说”强调NRTI磷酸化（活性部分）和NRTI三磷酸盐（-TP）对DNA pol γ （mtDNA复制酶）的抑制在获得性mtDNA耗竭的机制中。细胞激酶控制线粒体核苷酸和NRTI磷酸化。嘧啶nrti与dThd通过胸腺嘧啶激酶（细胞质TK1和线粒体TK2亚型）磷酸化为单磷酸盐（MP），通过胸腺嘧啶激酶（TMPK）磷酸化为二磷酸盐（DP）。工作假设是：野生型（WT） TK2或WT TMPK的转基因（TG）过表达通过增加线粒体dTTP质量和增加酶丰度来促进mtDNA复制。TMPK突变体（“功能增益”）对dTMP磷酸化有活性，但对AZTMP的活性增强。在缺乏AZT的情况下，这些TGs各自增加了dTTP的丰度。AZT治疗后，AZT- dp丰度显著增加，并被导入线粒体磷酸化为AZT- tp。相反，含有特定TK2点突变的TGs会降低TK2活性（“功能丧失”）。后者通过限制下游线粒体内加工的线粒体dTMP来消耗mtDNA。这里AZT处理通过AZT与dThd竞争TK2的磷酸化而增强mtDNA的消耗。线粒体AZT-TP与dTTP竞争通过DNA pol γ并入mtDNA。这抑制了mtDNA的复制，导致mtDNA的耗尽和突变，线粒体功能障碍和器官功能障碍。AIM 1使用具有心脏靶向、条件性、过表达WT TMPK、嵌合TMPK和突变TMPK的TGs定义nrti（如AZT） mtDNA缺失的分子、病理和生理事件。AIM 2定义了nrti（如AZT） mtDNA耗竭的分子、病理和生理事件，使用具有心脏靶向、条件、过表达WT TK2和突变TK2的TGs。实验为改进艾滋病的抗逆转录病毒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.