Mitochondrial G-quadruplex structures in health and disease

健康和疾病中的线粒体 G-四链体结构

• 批准号: 10221730
• 负责人: Brett A Kaufman
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221730
• 关键词: Acute; Address; Affect; Alleles; Antibodies; Berberine; Binding; Bioinformatics; Biology; Biophysics; Cell Line; Cell Respiration; Cells; Cellular Structures; ChIP-seq; Clinical; DNA Maintenance; DNA Sequence; DNA biosynthesis; Data; Development; Disease; Enzymes; Excision; Future; G-Quartets; Gene Expression; Genetic Transcription; Goals; Guanine; Health; Heritability; Intrabody; Knowledge; Lead; Ligands; Location; Malignant Neoplasms; Metabolic syndrome; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Matrix; Molecular; Nuclear; Nucleic Acids; Nucleotides; Pathogenicity; Pathologic; Pathology; Patient Care; Patients; Penetrance; Physiological; Ploidies; Property; Proteins; Publishing; Reagent; Regulation; Resolution; Respiration; Respiratory physiology; Role; Severities; Specificity; Structure; Testing; Therapeutic; Tissue Viability; Translations; Variant; Work; chromatin immunoprecipitation; early onset; fascinate; genome integrity; helicase; heteroplasmy; improved; innovation; mitochondrial genome; novel; novel strategies; respiratory; response; small molecule; tool

PROJECT SUMMARY G-quadruplex structures (G4) arise in guanine-rich sequences and have a high potential for formation in the mitochondrial DNA (mtDNA) due to its strand specific biases in nucleotide content. Preliminary studies and prior published work suggest that G4s impact mitochondrial function, but the evidence remains largely indirect, obscuring the role of these fascinating structures in normal and pathological mitochondrial biology. This proposal will address this gap in knowledge by defining the specific regions of mtDNA that form G4 in the cell and the conditions that promote G4 emergence and stability. The proposal is significant because a fuller understanding of the regulation of mtDNA maintenance and expression may be important in future approaches to diverse pathologies including heritable mitochondrial diseases, metabolic syndromes and sporadic cancers. The proposal is innovative in its development of novel reagents to detect mitochondrial G4s and in its novel approach to the therapy of mitochondrial disorders. The overarching hypothesis is that physiological G4 formation within mtDNA is widespread and regulates mitochondrial transcription and replication. The first specific aim will employ an innovative tool, a mitochondrial-targeted intrabody that binds to G4 sequences, to probe for G4-interacting sequences in the mitochondrial matrix by chromatin immunoprecipitation (ChIP). The sequences of mtG4s will be identified and their relative abundance will be evaluated under a range of conditions. The conditions include basal, elevated and inhibited mitochondrial function, and under G4-activated or G4-inhibited conditions. The role of DNA unwinding enzymes will also be evaluated. The second specific aim will expand upon a recent observation that induced G4 formation, using G4 binding agents, selects against specific pathogenic mtDNA variants that enhance G4 formation. Such variants typically exist in a state known as heteroplasmy, where healthy mtDNA is also present and the ratio between pathogenic and wild type sequence determines penetrance and severity. We will use patient cell lines and patient-derived cybrid cells to evaluate the range of pathogenic variants that may be susceptible to this approach. We will also expand the identification of novel G4 binding compounds that discriminate between pathogenic and wild type alleles. Overall, these studies will contribute mechanistic evidence for specific G4 structure formation in different conditions, connect their formation to the regulation of mtDNA replication and transcription, as well as develop new tools and reagent to positively impact mtDNA content in certain heteroplasmic conditions.

项目摘要 G-四链体结构（G4）以鸟嘌呤的序列出现，具有很高的形成潜力 线粒体DNA（mtDNA）由于其链核苷酸含量的链特异性偏置而引起的。初步研究和 事先发表的工作表明，G4S会影响线粒体功能，但证据在很大程度上是间接的， 掩盖了这些迷人的结构在正常和病理线粒体生物学中的作用。这 提案将通过定义形成细胞中G4的mtDNA的特定区域来解决知识的差距 以及促进G4出现和稳定性的条件。该提议很重要，因为更饱满 在将来的方法中，了解mtDNA维护和表达的调节可能很重要 对于包括遗传性线粒体疾病，代谢综合征和零星癌症的多种病理。 该提案在开发新型试剂的开发方面具有创新性，以检测线粒体G4及其小说 线粒体疾病治疗的方法。总体假设是生理G4 mtDNA内的形成是广泛的，并调节线粒体转录和复制。第一个 具体目标将采用创新工具，一种与G4序列结合的线粒体靶向的内在靶心内的工具 通过染色质免疫沉淀（CHIP）探测线粒体基质中G4相互作用序列的探针。这 将确定MTG4的序列，并将评估其相对丰度 状况。条件包括基础，升高和抑制线粒体功能，以及在G4激活下 或G4抑制条件。还将评估DNA解开酶的作用。第二个特定 AIM将在最近的观察结果上扩展，即使用G4结合剂诱导G4形成，选择反对 特定的致病mtDNA变体，增强了G4的形成。这种变体通常存在于已知状态 作为异质，也存在健康的mtDNA以及致病性和野生型之间的比率 序列决定了外观和严重程度。我们将使用患者细胞系和患者来源的Cybrid细胞 评估可能容易受到这种方法的致病变异范围。我们还将扩展 鉴定新型G4结合化合物，这些化合物区分了致病等位基因和野生型等位基因。 总体而言，这些研究将为不同的G4结构形成提供机械证据 条件，将其形成与mtDNA复制和转录的调节，并发展 在某些异质条件下，新工具和试剂可积极影响mtDNA含量。