Acquired mtDNA depletion and nucleoside reverse transciptase inhibitors

获得性 mtDNA 耗竭和核苷逆转录酶抑制剂

基本信息

批准号：
7910403
负责人：
WILLIAM LEWIS
金额：
$ 36.87万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7910403
关键词：
3&apos -azido-3&apos -deoxythymidine 5&apos phosphate AIDS/HIV problem Acquired Immunodeficiency Syndrome Address Adult Cardiac Cardiac Myocytes Cell physiology Chimera organism Coin DNA Defect Diphosphates Dose Enzymes Escherichia coli Event Exhibits Functional disorder Gene Expression Gene Targeting Genes Genetic Heart Highly Active Antiretroviral Therapy Homeostasis Link Methods Mitochondria Mitochondrial DNA Mitochondrial RNA Molecular Mono-S Mutation Myosin Heavy Chains Nucleosides Nucleotides Organ Oxidative Phosphorylation Pathogenesis Pathway interactions Phosphorylation Phosphotransferases Physiological Point Mutation Process Protein Isoforms Pyrimidine Pyrimidines Research Personnel Reverse Transcriptase Inhibitors Series Stavudine Stream TK1 gene TK2 gene Technology Testing Thymidine Kinase Time Toxic effect Transgenic Organisms Work Zidovudine gain of function heart cell improved in vivo inhibitor/antagonist insight loss of function mitochondrial DNA mutation mitochondrial dysfunction mutant overexpression polypeptide promoter replicase research study thymidylate kinase tool tripolyphosphate zidovudine triphosphate

项目摘要

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.

该项目定义了获得的线粒体的体内机制！（MT-）DNA耗竭由核苷引起 HIV/AIDS的逆转录酶抑制剂（NRTI）。由于嘧啶而改善了艾滋病毒/艾滋病的生存 Nrtis如Zidovudine（AZT）和Stavudine（D4T），但是NRTI线粒体毒性限制了治疗和mani- 节日为mtDNA耗竭和器官功能障碍。 “ DNA pol y假设”强调了NRTI磷酸 NRTI三磷酸盐（-tp）中对DNA pol y（mtDNA复制酶）的抑制（-tp）在中获得的mtDNA耗竭的机制。细胞激酶控制线粒体核苷酸和NRTI 磷酸化。嘧啶NRTI与DTHD竞争，以磷酸化对单磷酸盐（-MP）通过胸苷激酶（细胞质TK1和线粒体TK2同工型）和双磷酸盐（-DP）通过胸苷激酶（TMPK）。工作假设状态：野生型的转基因（TG）过表达（WT）TK2或WT TMPK通过在平行的线粒体DTTP质量中增加MTDNA复制随着酶丰度的增加。具有TMPK突变体（“功能的获得”）的TGS具有DTMP的活性磷酸化，但用AZTMP表现出增强的活性。在没有AZT的情况下，这些TG都会增加 DTTP丰度。通过AZT治疗，AZT-DP的丰度大大增加，并进口到Mito- 用于磷酸化AZT-TP的软骨。相反，具有特定TK2点突变的TGS减少 TK2活动（“功能丧失”）。这些后一种TGS通过限制线粒体DTMP来耗尽mtDNA 流中线粒体处理。这里的AZT治疗可以通过AZT的竞争增强mtDNA耗竭用TK2进行DTHD进行磷酸化。线粒体AZT-TP与DTTP竞争，将 MTDNA由DNA pol y。这抑制mtDNA复制，导致mtDNA耗竭和突变，线粒体功能障碍和器官功能障碍。 AIM 1定义了NRTIS的mtDNA耗竭中的分子，病理和生理事件（如AZT）使用带有心脏靶向的，有条件的，WT TMPK，嵌合TMPK和突变体的TGS TMPK。 AIM 2定义了MtDNA耗竭的分子，病理和生理事件（如 AZT）使用TGS与心脏靶向，有条件的，过表达WT TK2和突变体TK2。实验提供了改善用于艾滋病抗逆转录病毒的治疗的见解。