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

基本信息

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
项目类别：
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

项目摘要

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，每个基因组分为细胞核和线粒体。双基因组细胞该系统是通过早期内共生细菌基因组和原核细菌的共同进化而建立的我们祖先细胞十亿年来的基因组。线粒体和线粒体反映了它们长期而密切的关系细胞核之间积极沟通以协调各种细胞功能。这样的线粒体核通信对于细胞健康和衰老至关重要，并且越来越被认为是高度复杂和可靠的复杂的。然而，虽然超过 1,000 个核编码蛋白直接调节线粒体，但没有线粒体- 已知编码因子可以主动调节细胞核。我们最近发布了一流的调节核基因组的线粒体编码肽（即 MOTS-c）。在此，我们向大家介绍一个未发表的与 MOTS-c 有遗传关联的新型线粒体编码基因，我们将其命名为 MOTS-b。值得注意的是，MOTS- b 和 MOTS-c 在细胞核中相互作用，由免疫共沉淀偶联蛋白质组学确定（质谱）。与 MOTS-c 一样，MOTS-b 的核易位似乎是作为一种适应性调节的回复。例如，MOTS-b 和 MOTS-c 都在时间上动态易位到细胞核。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，并且可以根据我们的体外进化研究直接结合 DNA，以识别特定的 MOTS-b 靶向核苷酸序列。在功能层面，MOTS-b治疗调节细胞增殖和代谢，这又与 MOTS-c 一致。在这里，我们建议描述和验证 MOTS-b 的核作用。本研究的总体假设提议认为 MOTS-b 是一种新型线粒体编码基因，可易位至细胞核并直接与 MOTS-c 协调调节适应性基因表达。首先，我们将表征分子和使用多管齐下的方法研究 MOTS-b 的细胞机制，包括诱变、免疫共沉淀、邻近标记辅助蛋白质组学和 DNA 结合测定。我们将绘制功能肽结构域 MOTS-b 与 DNA 结合和肽相互作用有关，我们假设这对其核重要角色。我们还将确定触发 MOTS-b 易位至细胞核的细胞环境/事件。然后，我们将通过有压力和无压力的全局无偏 RNA-seq 来确定 MOTS-b 诱导的转录组，这将通过 MOTS-b 结合染色质位点的全基因组图谱 (ChIP-seq) 进行补充，这些位点位于开放区域内染色质（ATAC-seq）。然后我们将筛选介导 MOTS-b 对细胞影响的候选基因增殖和代谢。了解线粒体基因组中编码的调节因子的贡献将提供更多信息具有附加生物学意义的全面基因组视角。如果成功，我们预测我们的研究将通过识别线粒体编码的新家族，对（i）衰老的基础生物学产生广泛而持久的影响核基因组的调节因子，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）转化发展将 mtDNA 描述为新型治疗/诊断靶点的来源。