Acquired mtDNA depletion and nucleoside reverse transciptase inhibitors

这个项目定义了获得性线粒体的体内机制！核苷引起的(MT-)DNA耗竭 艾滋病毒/艾滋病的逆转录酶抑制剂(NRTI)。由于嘧啶，艾滋病毒/艾滋病患者的存活率有所提高 NRTI如齐多夫定(AZT)和司他夫定(D4T)，但NRTI的线粒体毒性限制了治疗和治疗。 表现为线粒体DNA耗尽和器官功能障碍。“DNA Poly假说”强调了NRTI荧光体-- NRTI三磷酸(-TP)对DNA Poly(线粒体DNA复制酶)的活化和抑制作用 获得性线粒体DNA耗竭的机制。细胞激酶控制线粒体核苷酸和NRTI 磷酸化。嘧啶核糖核酸二异构体与dThd竞争磷酸化为一磷酸 胸苷激酶(细胞质TK1和线粒体TK2亚型)和二磷酸(-DP)通过 胸苷酸激酶(TMPK)。工作假说：转基因(TG)野生型过表达 (WT)TK2或WT TMPK通过平行增加线粒体dTTP质量促进mtDNA复制 随着酶丰度的增加。带有TMPK突变体(“功能增益”)的TGS与DTMP结合是有效的 磷酸化，但与AZTMP一起显示出增强的活性。在没有AZT的情况下，这些TGS各自增加 DTTP丰度。使用AZT治疗后，AZT-DP的丰度急剧增加，并被导入有丝分裂细胞。 线粒体对AZT-TP的磷酸化作用。相反，携带特定TK2点突变的TGS减少 TK2活性(“功能丧失”)。后一种TGS通过限制线粒体DTMP向下延伸来耗尽mtDNA。 流式线粒体内处理。AZT治疗通过AZT的竞争增强mtDNA耗竭 与dThd结合用于TK2的磷酸化。线粒体AZT-TP与dTTP竞争掺入 这抑制了mtDNA的复制，导致mtDNA枯竭和突变，线粒体 功能障碍和器官功能障碍。 目的1明确NRTIs(如AZT)线粒体DNA耗竭的分子、病理和生理事件 将TGS用于心脏靶向、条件性、WT TMPK、嵌合TMPK和突变体的过表达 TMPK。目标2定义NRTIs(如)线粒体DNA耗尽的分子、病理和生理事件 AZT)使用心脏靶向的、有条件的、过表达WT TK2和突变体TK2的TGS。 实验为改进艾滋病抗逆转录病毒药物的治疗提供了见解。

This project defines in vivo mechanisms of acquired mitochondria! (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 mani- fests as mtDNA depletion and organ dysfunction. The "DNA pol y hypothesis" underscores NRTI phosphor- ylation (to active moieties) and inhibition of DNA pol y (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 mito- chondria 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 y. 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, overexpressionof WT TK2 and mutant TK2. Experiments offer insights into improving therapy with antiretrovirals used in AIDS.