Mitochondrial-Encoded Regulators of the Nucleus and Cellular Homeostasis

线粒体编码的细胞核和细胞稳态调节因子

• 批准号: 10527988
• 负责人: Changhan Lee
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527988
• 关键词: ATAC-seq; Aging; Bacterial Genome; Base Sequence; Binding; Biological; Biological Assay; Biological Process; Biology of Aging; CRISPR/Cas technology; Candidate Disease Gene; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; ChIP-seq; Chromatin; Circular DNA; Co-Immunoprecipitations; Communication; Complement; Complex; Computer Analysis; Confocal Microscopy; Coupled; DNA; DNA Binding; DNA Sequence; Data; Dermal; Development; Diagnostic; Disease; Dose; Doxorubicin; Event; Evolution; Exercise; Family; Fibroblasts; Gel; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genome; Genomic approach; Genomics; Growth; Homeostasis; Human; In Vitro; Individual; Knock-out; Label; Ligands; Link; Machine Learning; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Mitochondria; Mitochondrial DNA; Molecular; Mutagenesis; Names; Nuclear; Nuclear Translocation; Nucleotides; Open Reading Frames; Organelles; Pathway interactions; Pattern; Peptides; Proteins; Proteomics; Publishing; RNA, ribosomal, 12S; Regulation; Reporting; Reproduction; Respiration; Ribosomal RNA; Role; Sampling; Scheme; Serum; Site; Source; Stains; Starvation; Statistical Data Interpretation; Stress; System; Tertiary Protein Structure; Testing; Validation; age related; base; biological adaptation to stress; design; fitness; functional genomics; genome-wide; healthy aging; knock-down; mitochondrial genome; monocyte; neuronal cell body; novel; novel therapeutics; peptide I; repaired; response; small hairpin RNA; transcriptome; transcriptome sequencing

ABSTRACT Our cells have two genomes, each compartmentalized in the nucleus and mitochondria. The bi-genomic cellular system was established through co-evolution of the early endosymbiotic bacterial genome and the proto-nuclear genome of our ancestral cell over a billion years. Reflecting their long and close relationship, mitochondria and the nucleus actively communicate with each other to coordinate various cellular functions. Such mitonuclear communication is vital to cellular fitness and aging, and increasingly appreciated to be highly sophisticated and complex. However, whereas >1,000 nuclear-encoded proteins directly regulate the mitochondria, no mitochondrial- encoded factors have been known to actively regulate the nucleus. We recently published the first-in-class mitochondrial-encoded peptide (i.e. MOTS-c) that regulates the nuclear genome. Here, we present an unpublished novel mitochondrial-encoded gene that is genetically linked to MOTS-c, which we named MOTS-b. Notably, MOTS- b and MOTS-c interact with each other in the nucleus, determined by co-immunoprecipitation-coupled proteomics (mass spectrometry). Like MOTS-c, the nuclear translocation of MOTS-b appears to be regulated as an adaptive response. For instance, MOTS-b and MOTS-c both dynamically translocate to the nucleus in a temporally coordinated manner upon monocyte differentiation. Further, MOTS-b is enriched in purified nuclear chromatin samples and can directly bind DNA based on our in vitro evolution studies to identify specificMOTS-b-targeted nucleotide sequences. At the functional level, MOTS-b treatment regulates cellular proliferation and metabolism, which again is consistent with MOTS-c. Here, we propose to characterize and validate the nuclear role of MOTS-b. The overarching hypothesis of this proposal is that MOTS-b is a novel mitochondrial-encoded gene that translocates to the nucleus and directly regulates adaptive gene expression in coordination with MOTS-c. First, we will characterize the molecular and cellular mechanisms of MOTS-b using a multipronged approach including mutagenesis, co-immunoprecipitation, proximity-labeling assisted proteomics, and DNA-binding assays. We will map the functional peptide domains of MOTS-b pertinent to DNA binding and peptide interaction, which we hypothesize to be important for its nuclear role. We will also determine the cellular context/event that triggers MOTS-b to translocate to the nucleus. Then, we will determine the MOTS-b-induced transcriptome by global unbiased RNA-seq with and without stress, which will be complemented by genome-wide mapping of MOTS-b-bound chromatin sites (ChIP-seq) that are within open chromatin (ATAC-seq). We will then screen candidate genes that mediate the effects of MOTS-b on cellular proliferation and metabolism. Understanding the contributions of regulators encoded in the mitochondrial genome will provide a more comprehensive genomic perspective with added biological significance. If successful, we predict that our study will have broad and lasting impact on (i) basic biology of aging by identifying a novel family of mitochondrial-encoded regulators of the nuclear genome, which provides another layer of complexity to gene regulation in our co-evolved bi-genomic cellular system and their role in aging and age-related diseases, and (ii) translational development by describing the mtDNA as a source of novel therapeutic/diagnostic targets.

抽象的 我们的细胞有两个基因组，每个基因组都在细胞核和线粒体中划分。双基因组细胞 通过早期内共生细菌基因组和原始核的共同进化来建立系统 我们祖先细胞的基因组十亿年。反映了他们长期而亲密的关系，线粒体和 核彼此积极通信以协调各种细胞功能。这样的有线核 沟通对于细胞健身和衰老至关重要，并且越来越多地赞赏高度复杂，并且 复杂的。但是，>> 1,000个核编码蛋白直接调节线粒体，但没有线粒体 已知编码因子积极调节核。我们最近出版了第一类 线粒体编码的肽（即MOTS-C）调节核基因组。在这里，我们提出了未出版的 新型线粒体编码的基因与MOTS-C有遗传有关，我们将其命名为MOTS-B。值得注意的是，mot- B和MOTS-C在核中相互相互作用，由共免疫沉淀耦合蛋白质组学确定 （质谱法）。像MOTS-C一样，MOTS-B的核易位似乎是自适应的 回复。例如，MOTS-B和MOTS-C都动态转移到时间上的核 单核细胞分化时的协调方式。此外，MOTS-B富含纯化的核染色质 样品并可以基于我们的体外进化研究直接结合DNA，以识别以B靶向的特定MOTS-B 核苷酸序列。在功能水平上，MOTS-B处理调节细胞增殖和代谢， 这再次与MOTS-C一致。 在这里，我们建议表征和验证MOTS-B的核作用。总体假设 建议是MOTS-B是一种新型的线粒体编码基因，可转移到细胞核并直接 调节与MOTS-C协调中的适应性基因表达。首先，我们将表征分子和 MOTS-B的细胞机制，使用多血管的方法，包括诱变，共免疫沉淀， 接近标记的辅助蛋白质组学和DNA结合测定法。我们将映射 MOTS-B与DNA结合和肽相互作用有关，我们假设这对于其核很重要 角色。我们还将确定触发MOTS-B以转移到细胞核的细胞上下文/事件。然后， 我们将通过有或没有压力的全局无偏的RNA-seq来确定MOTS-B诱导的转录组，这 将通过在开放端的MOTS-B结合染色质位点（CHIP-SEQ）的全基因组映射来补充 染色质（ATAC-SEQ）。然后，我们将筛选介导MOTS-B对细胞影响的候选基因 扩散和代谢。 了解线粒体基因组中编码的调节剂的贡献将提供更多 全面的基因组观点具有增加的生物学意义。如果成功，我们预测我们的研究将 通过识别一个新的线粒体编码家庭，对（i）对（i）衰老的基本生物学产生了广泛的影响 核基因组的调节剂，该基因组为我们共同进化的基因调节提供了另一层复杂性 双基因组细胞系统及其在衰老和年龄相关疾病中的作用，以及（ii）通过 将mtDNA描述为新的治疗/诊断靶标的来源。