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: 10434152
• 负责人: Zengyi Shao
• 金额: $ 36.8万
• 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/10434152
• 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; Foundations; 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.

项目总结/摘要 爱荷华州州立大学的Shao小组旨在利用他们在遗传工具开发方面的专业知识， 组装一个全面的线粒体基因工具包。线粒体DNA的致病作用是牵连 在无数人类疾病和失调中的作用。然而，人类线粒体功能障碍的研究遭受 缺乏理想的真核生物模型。研究较多的模式酵母S.酿酒酵母，由于其 线粒体生理学与人类相差太远。我们建议开发一种新的有前途的模型， 提高了对人类线粒体DNA的认识，对线粒体功能障碍和致病性产生了新的认识。 我们已经确定解脂耶氏酵母在实用性（即，低成本和 作为低真核生物的遗传操作的快速时间尺度）和易处理性（即，反映了人类的义务 有氧需要）。此外，目前绝大多数线粒体功能障碍研究仅关注 核编码线粒体基因异常这主要是由于爆炸物 核基因组编辑技术在“后CRISPR”时代的进展尚未转化为 mtDNA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 策略被证明是有效的，我们设想许多mtDNA操作工具将由研究实验室开发 围绕着地球仪，沿着与CRISPR核基因组操作革命相同的轨迹。 在接下来的五年里，除了基础工具的开发，我们将努力阐明 新模型中的mtDNA-表型关系。Y.脂肪分解，积累脂质，使其成为一个 特别适合于人脂肪细胞的模型。脂肪组织中的线粒体功能障碍涉及一种 危害人类健康的广泛流行病。通过我们的线粒体遗传学的发展 工具包，我们将以精确的方式重建mtDNA相关的人类病理， 发展和所有后续的机制研究。此外，我们建议研究 调节线粒体内膜的流动性，改变线粒体生理学， 在未来发现与肥胖有关的疾病的潜在治疗方法。最后，沿着沿着 研究分支，我们将整合开发的线粒体遗传工具包与我们以前的努力， 代谢工程我们将利用单个细胞中线粒体的多样性以及高拷贝 在一个单一的线粒体mtDNA的数量，以提高剂量的基因编码的限速步骤， 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， 产生多种衍生自TCA循环的化合物或其生物合成机制需要 高ATP输入。总之，这个MIRA的ESI项目将使我能够进入研究领域， 我现有的，并给予我的灵活性，以遵循重要的新的研究方向的机会出现。