Acquired mtDNA depletion and nucleoside reverse transciptase inhibitors

该项目定义了获得性线粒体的体内机制！(mt-)核苷引起的DNA缺失 逆转录酶抑制剂（NRTI）用于HIV/AIDS。嘧啶改善了艾滋病毒/艾滋病患者的生存率 NRTI如齐多夫定（AZT）和司他夫定（d4 T），但NRTI线粒体毒性限制了治疗和治疗。 线粒体DNA缺失和器官功能障碍。“DNA聚合假说”强调了NRTI磷光体- NRTI三磷酸盐（-TP）对DNA聚合酶（mtDNA复制酶）的作用和抑制作用， 获得性mtDNA缺失的机制。细胞激酶控制线粒体核苷酸和NRTI 磷酸化嘧啶NRTI与dThd竞争磷酸化为单磷酸（-MP）， 胸苷激酶（细胞质TK 1和线粒体TK 2亚型）和二磷酸（-DP）， 胸苷酸激酶（TMPK）。工作假设指出：野生型的转基因（TG）过表达 (WT)TK 2或WT TMPK通过平行增加线粒体dTTP质量促进mtDNA复制 增加酶的丰度。具有TMPK突变体（“功能获得”）的TG对dTMP具有活性 磷酸化，但与AZTMP表现出增强的活性。在没有AZT的情况下，这些TG各自增加 dTTP丰度。随着AZT治疗，AZT-DP丰度急剧增加，并输入到Mito- AZT-TP的磷酸化。相反，携带特异性TK 2点突变的TG减少， TK 2活性（“功能丧失”）。这些后一种TG通过限制线粒体dTMP来减少线粒体DNA， 流线粒体内加工。AZT治疗通过AZT的竞争增强线粒体DNA消耗 用dThd进行TK 2磷酸化。线粒体AZT-TP与dTTP竞争掺入 线粒体DNA的DNA聚合。这会抑制mtDNA复制，导致mtDNA缺失和突变， 以及器官功能障碍。 AIM 1定义了NRTI（如AZT）中mtDNA缺失的分子、病理和生理事件 使用具有心脏靶向的、条件性的WT TMPK、嵌合TMPK和突变TMPK过表达的TG TMPK。AIM 2定义了NRTI（如 AZT），使用具有心脏靶向的、条件性的WT TK 2和突变TK 2的过表达的TG。 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。

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.