The Role of Phosphate Manganese and Iron on Eukaryotic Oxidative Stress

磷酸锰和铁对真核氧化应激的作用

• 批准号: 7912197
• 负责人: Amit Ram Reddi
• 金额: $ 5.05万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912197
• 关键词: Aging; Air; Animal Model; Antioxidants; Binding; Biological Availability; Cardiovascular Diseases; Cells; Cuprozinc Superoxide Dismutase; Disease; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Engineering; Enzymes; Exhibits; Genes; Genetic Screening; Heart; Human; Injury; Investigation; Ions; Iron; Libraries; Life; Link; Malignant Neoplasms; Manganese; Mediating; Metabolism; Monitor; Nature; Neurologic; Oxidative Stress; Oxygen; Play; Polyphosphates; Polyps; Proteins; Reactive Oxygen Species; Reperfusion Injury; Reporting; Research Design; Resistance; Role; Saccharomyces cerevisiae; Series; Severities; Spectrum Analysis; Staining method; Stains; Stress; Superoxide Dismutase; Superoxides; Testing; Toxic effect; Variant; Yeasts; design; human disease; inorganic phosphate; insight; manganese phosphate; mutant; oxidative damage; public health relevance; repaired; research study; small molecule; therapeutic development

DESCRIPTION (provided by applicant): Intracellular manganese ions (Mn) and the enzyme Cu/Zn superoxide dismutase (SOD1) have overlapping roles in oxidative stress protection. While the mechanism of SOD1 action in superoxide detoxification has been well characterized, very little is understood about how cells utilize Mn to suppress oxidative damage independent of SOD enzymes. Recently, using S. cerevisiae as a model organism, we have reported that proper phosphate metabolism is important for suppressing oxidative damage and critical for enabling cells to utilize Mn as an antioxidant. It was found that sod1 null stains engineered to hyperaccumulate phosphate are oxidatively stressed and inviable in air. Preliminary results indicate that high cytoplasmic polyphosphate (PolyP) is responsible for the severity of oxidative damage and phosphate interactions with both Mn and Fe are involved. We hypothesize that PolyP enhances oxidative injury by sequestering Mn and Fe, thereby limiting their availability to the Mn-antioxidant and to essential Fe/S proteins that are susceptible to oxidative injury. The purpose of the current proposal is to test this hypothesis and elucidate the nature of the Mn-antioxidant. In order to determine the role of PolyP in oxidative stress, a series of yeast strains that have altered PolyP metabolism will be engineered. These strains, hereafter referred to as the polyphosphate titratable series (PTS), which will have variations in the size, content, and cellular localization of PolyP, will be exploited to assess the impact of PolyP on various indicators of oxidative stress and on Mn and Fe bioavailability. In the sod1 null background, the PTS strains can be used to determine how PolyP influences Mn-suppression of oxidative damage and Fe availability for repairing damaged Fe/S clusters. Furthermore, we will directly monitor Mn- and Fe-PolyP interactions inside the PTS mutants as a function of oxidative stress resistance by using a newly developed application of ENDOR spectroscopy to whole cells. In toto, these experiments will reveal exactly how polyphosphate influences oxidative stress and the role Mn and Fe play in mediating its toxicity. In addition, the mechanism of Mn suppression of oxidative stress will be determined by employing a high-throughput genetic screen to identify genes that are required for Mn-antioxidant activity. sod1 null yeast will be mutagenized with a transposon library and mutants that exhibit loss of Mn rescue of oxidative damage will be selected. This screen is designed to select for genes that are involved in the metabolism of small molecules that bind and activate Mn for Mn-antioxidant activity. Overall, these studies should provide great insight into the role of phosphate, Mn, and Fe in cellular oxidative stress and the factors that govern Mn suppression of oxidative damage. Studies of this type are at the heart of understanding and perhaps treating the numerous human disorders attributed to oxidative stress. PUBLIC HEALTH RELEVANCE: Damage from oxygen radicals has been linked to a number of human diseases, including reperfusion injury, cancer, cardiovascular disease, neurological degeneration, and aging. Studies into the basic mechanisms of cellular oxidative stress resistance are crucial towards understanding the role of oxygen radicals in disease and to the eventual development of therapeutic strategies. We have recently shown that, in addition to superoxide dismutase (SOD) enzymes, manganese is critical for sustaining life in atmospheric oxygen. However, very little is understood about how cells utilize Mn as an antioxidant. The purpose of the current investigation is to decipher the mechanism of cellular Mn-antioxidant activity, with a particular emphasis on the interplay between phosphate, Mn and Fe metabolism.

描述（由申请人提供）：细胞内锰离子（Mn）和铜/锌超氧化物歧化酶（SOD1）在氧化应激保护中具有重叠的作用。虽然 SOD1 在超氧化物解毒中的作用机制已得到很好的表征，但人们对细胞如何利用 Mn 来抑制独立于 SOD 酶的氧化损伤知之甚少。最近，我们使用酿酒酵母作为模式生物，报道了适当的磷酸盐代谢对于抑制氧化损伤非常重要，并且对于使细胞能够利用锰作为抗氧化剂至关重要。研究发现，设计用于超积累磷酸盐的 sod1 无效染色剂受到氧化应激，并且在空气中无法存活。初步结果表明，高细胞质多磷酸盐 (PolyP) 是造成严重氧化损伤的原因，并且磷酸盐与 Mn 和 Fe 的相互作用也参与其中。我们假设 PolyP 通过隔离 Mn 和 Fe 来增强氧化损伤，从而限制它们对 Mn 抗氧化剂和易受氧化损伤影响的必需 Fe/S 蛋白的可用性。当前提案的目的是检验这一假设并阐明锰抗氧化剂的性质。为了确定 PolyP 在氧化应激中的作用，将设计一系列改变 PolyP 代谢的酵母菌株。这些菌株（以下称为聚磷酸盐可滴定系列 (PTS)）在 PolyP 的大小、含量和细胞定位方面存在差异，将用于评估 PolyP 对各种氧化应激指标以及对 Mn 和 Mn 的影响。铁的生物利用度。在 sod1 无效背景下，PTS 菌株可用于确定 PolyP 如何影响 Mn 对氧化损伤的抑制以及修复受损 Fe/S 簇的 Fe 可用性。此外，我们将通过使用新开发的 ENDOR 光谱对全细胞的应用，直接监测 PTS 突变体内部 Mn-和 Fe-PolyP 相互作用作为氧化应激抗性的函数。总而言之，这些实验将准确揭示多磷酸盐如何影响氧化应激以及锰和铁在介导其毒性中所起的作用。此外，通过采用高通量遗传筛选来鉴定锰抗氧化活性所需的基因，可以确定锰抑制氧化应激的机制。 sod1无效酵母将用转座子文库进行诱变，并且将选择表现出Mn缺失拯救氧化损伤的突变体。该筛选旨在选择参与小分子代谢的基因，这些小分子结合并激活锰以实现锰抗氧化活性。总的来说，这些研究应该可以深入了解磷酸盐、锰和铁在细胞氧化应激中的作用以及控制锰抑制氧化损伤的因素。此类研究是理解并可能治疗由氧化应激引起的众多人类疾病的核心。 公众健康相关性：氧自由基造成的损害与许多人类疾病有关，包括再灌注损伤、癌症、心血管疾病、神经退行性变和衰老。研究细胞氧化应激抵抗的基本机制对于了解氧自由基在疾病中的作用以及最终制定治疗策略至关重要。我们最近发现，除了超氧化物歧化酶（SOD）之外，锰对于在大气中的氧气中维持生命也至关重要。然而，人们对细胞如何利用锰作为抗氧化剂知之甚少。当前研究的目的是破译细胞锰抗氧化活性的机制，特别强调磷酸盐、锰和铁代谢之间的相互作用。