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: 10428535
• 负责人: Brett A Kaufman
• 金额: $ 36.28万
• 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/10428535
• 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.

项目总结 G-四链结构(G4)出现在富含鸟嘌呤的序列中，并在 线粒体DNA(MtDNA)由于其在核苷酸含量上的链特异性偏向。初步研究和 先前发表的研究表明，G4S影响线粒体功能，但证据主要是间接的， 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 一项提案将通过定义细胞中形成G4的线粒体DNA的特定区域来解决这一知识差距 以及促进G4出现和稳定的条件。这项提议意义重大，因为更完整的 了解线粒体DNA维持和表达的调节在未来的方法中可能是重要的 不同的病理类型，包括遗传性线粒体疾病、代谢综合征和散发性癌症。 该提案在开发检测线粒体G4S的新型试剂方面具有创新性，并在其新颖的 线粒体疾病的治疗探讨。最重要的假设是生理性的G4 线粒体DNA中的形成广泛存在，并调节线粒体的转录和复制。第一 特定的目标将使用一种创新的工具，即结合G4序列的线粒体靶向体内，以 用染色质免疫沉淀(CHIP)检测线粒体基质中G4相互作用序列。这个 将确定mtG4的序列，并将在以下范围内评估它们的相对丰度 条件。这些情况包括基础、升高和抑制的线粒体功能，以及在G4激活的情况下 或G4抑制的条件。DNA解旋酶的作用也将得到评估。第二个具体问题 AIM将扩展最近的一项观察，即使用G4结合剂诱导G4的形成，选择反对 增强G4形成的特定致病线粒体DNA变体。这些变体通常以已知的状态存在 作为异质性，也存在健康的mtdna，以及致病性和野生型之间的比率 顺序决定了外显性和严重性。我们将使用患者细胞系和患者来源的胞质细胞来 评估可能对这种方法敏感的致病变异的范围。我们还将扩大 区分致病和野生型等位基因的新型G4结合化合物的鉴定。 总体而言，这些研究将为不同地区特定的G4结构的形成提供力学证据 它们的形成与mtdna复制和转录的调节以及发育有关。 在某些异质性条件下积极影响线粒体DNA含量的新工具和试剂。