Establishing a Novel Instrumental Model for Elucidating Mitochondrial DNA-Associated Dysfunction and Pathogenicity

建立阐明线粒体 DNA 相关功能障碍和致病性的新仪器模型

• 批准号: 10622629
• 负责人: Zengyi Shao
• 金额: $ 36.74万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622629
• 关键词: Adipocytes; Adipose tissue; Aerobic; Area; Biochemical Pathway; Biology; Cells; Citric Acid Cycle; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Disease; Epidemic; Equilibrium; Eukaryota; Functional disorder; Future; Gene Abnormality; Gene Dosage; Genetic; Genome; Grant; Health; Human; Human Pathology; Inner mitochondrial membrane; Iowa; Knowledge; Lipids; Mediating; Mitochondria; Mitochondrial DNA; Modeling; Nuclear; Nucleic Acids; Obesity associated disease; Pathogenicity; Phenotype; Physiology; RNA; Research; Role; Saccharomyces cerevisiae; Side; Study models; Technology; Therapeutic; Translating; Universities; Yarrowia lipolytica; Yeast Model System; cost; drug development; engineering design; flexibility; fluidity; genetic manipulation; genome editing; human disease; insight; metabolic engineering; mitochondrial dysfunction; novel; obesity treatment; reconstitution; stem; tool; tool development

Project Summary/Abstract The Shao group at Iowa State University aims to leverage their expertise in genetic tool development to assemble a comprehensive mitochondrial genetic toolkit. The causative role of mitochondrial DNA is implicated in a myriad of human diseases and disorders. However, human mitochondrial dysfunction studies suffer from the absence of an ideal eukaryotic model. The well-studied model yeast, S. cerevisiae, is unsuitable due to its mitochondrial physiology deviating too far from humans. We propose developing a new promising model to heighten knowledge of human mtDNA, yielding new insights on mitochondrial dysfunction and pathogenicity. We have identified that Yarrowia lipolytica strikes a perfect balance between practicality (i.e., low cost and quick timescale of genetic manipulations as a low eukaryote) and tractability (i.e., mirroring human's obligate aerobic needs). In addition, the overwhelming majority of current mitochondrial dysfunction studies focus solely on nuclear-encoded mitochondrial gene abnormalities. This is mainly attributed to the fact that the explosive progression of nuclear genome editing technology in the epoch of “post-CRISPR” has yet to translate to mtDNA editing. We propose leveraging a recently discovered stem-loop RNA motif to overcome nucleic acid import limitations – the largest technical barrier to the development of CRISPR-associated technologies. If this strategy proves effective, we envision that many mtDNA manipulation tools will be developed by research labs around the globe, following the same trajectory as the CRISPR nuclear genome manipulation revolution. Over the next five years, in addition to the foundational tool development, we will strive for elucidating mtDNA-phenotype relationships in the new model. The ability of Y. lipolytica to accumulate lipids makes it a particularly suitable model for human adipocytes. Mitochondrial dysfunction in adipose tissue is involved in a broad spectrum of epidemics plaguing human health. Mediated by our development of a mitochondrial genetic toolkit, we will reconstitute mtDNA-associated human pathologies in a precise manner, enabling tailored drug development and all the subsequent mechanistic studies. Moreover, we propose studying the impact of modulating the fluidity of the inner mitochondrial membrane on altering mitochondrial physiology, which will lead to the discovery of potential treatments of obesity-related diseases in the future. Lastly, along a side research branch, we will integrate the developed mitochondrial genetic toolkit with our previous efforts in metabolic engineering. We will leverage the multiplicity of mitochondria in a single cell as well as the high copy number of mtDNA in a single mitochondrion to boost the dosage of the gene encoding the rate-limiting step in a biochemical pathway. This strategy will revolutionize the metabolic engineering design of eukaryotic hosts to produce a wide variety of compounds derived from TCA cycle or whose biosynthetic mechanism requires a high ATP input. Altogether, this MIRA for ESI project will enable me to move into research areas distinct from my existing ones and grant me the flexibility to follow important new research directions as opportunities arise.

项目摘要/摘要 爱荷华州立大学的Shao小组旨在利用他们在遗传工具开发方面的专业知识来 组装一个全面的线粒体遗传工具包。线粒体DNA的致病作用是牵连的 在无数的人类疾病和无序中。然而，人类线粒体功能障碍的研究受到 缺乏理想的真核细胞模型。研究得很好的模型酵母，酿酒酵母，不适合，因为它 线粒体生理学偏离人类太远。我们建议开发一种新的有前途的模式来 提高对人类线粒体DNA的认识，对线粒体功能障碍和致病性产生新的见解。 我们已经确认，解脂亚罗维菌在实用性(即低成本和 作为低等真核生物的遗传操作的快速时间表)和可驯化(即，反映人类的专有 有氧需求)。此外，目前绝大多数线粒体功能障碍的研究仅集中于 核编码的线粒体基因异常。这主要是因为炸药 后CRISPR时代的核基因组编辑技术的进展尚未转化为 线粒体DNA编辑。我们建议利用最近发现的茎环RNA基序来克服核酸 进口限制--CRISPR相关技术开发的最大技术障碍。如果这个 策略被证明是有效的，我们预计许多线粒体DNA操作工具将由研究实验室开发 在全球范围内，遵循与CRISPR核基因组操作革命相同的轨迹。 在未来五年中，除了基础工具的开发外，我们还将努力阐明 线粒体DNA与新模型的表型关系。解脂耶尔森氏菌积累脂质的能力使其成为一种 特别适合人脂肪细胞模型。脂肪组织中的线粒体功能障碍与 广泛的流行病困扰着人类健康。由我们的线粒体基因的发展所介导的 工具包，我们将以精确的方式重建与线粒体DNA相关的人类病理，使定制的药物成为可能 发展和所有随后的机械论研究。此外，我们建议研究 通过调节线粒体内膜的流动性来改变线粒体的生理学，这将 从而在未来发现肥胖相关疾病的潜在治疗方法。最后，沿着一个侧面 研究分支，我们将把开发的线粒体遗传工具箱与我们之前在 代谢工程学。我们将利用单个细胞中线粒体的多样性以及高拷贝 单个线粒体中mtDNA的数量，以增加编码限速步骤的基因的剂量 一种生化途径。这一策略将彻底改变真核宿主的代谢工程设计 产生从TCA循环衍生的各种化合物，或者其生物合成机制需要 高ATP投入。总之，这个Mira for ESI项目将使我能够进入不同于 并给予我灵活性，使我能够在机会出现时遵循重要的新研究方向。