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: 8053338
• 负责人: Amit Ram Reddi
• 金额: $ 1.52万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8053338
• 关键词: Aconitate Hydratase; Aerobic; Aging; Air; Animal Model; Antioxidants; Binding; Biochemical; Biological Availability; Cardiovascular Diseases; Cells; Cellular biology; Cuprozinc Superoxide Dismutase; Disease; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Engineering; Enzymes; Eukaryotic Cell; Exhibits; Generations; Genes; Genetic; Genetic Screening; Growth; Health; Heart; Human; Hydroxyl Radical; Injury; Investigation; Ions; Iron; Libraries; Life; Link; Malignant Neoplasms; Manganese; Mediating; Metabolism; Metals; Monitor; Nature; Neurologic; Organism; Oxidation-Reduction; Oxidative Stress; Oxygen; Play; Polyphosphates; Production; Protein S; Proteins; Reactive Oxygen Species; Reperfusion Injury; Reporting; Research Design; Resistance; Role; Saccharomyces cerevisiae; Series; Severities; Spectrum Analysis; Staining method; Stains; Starvation; Stress; Superoxide Dismutase; Superoxides; Testing; Toxic effect; Variant; Work; Yeasts; arginase; base; cofactor; design; human disease; inorganic phosphate; insight; manganese phosphate; mutant; null mutation; oxidation; oxidative damage; prevent; repaired; research study; response; small molecule; therapeutic development; uptake

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.

描述（由申请人提供）：细胞内锰离子（MN）和酶Cu/Zn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理已经很好地表征了，但关于细胞如何利用MN抑制独立于SOD酶的氧化损伤的理解很少。最近，使用酿酒酵母作为模型生物，我们报告说，适当的磷酸代谢对于抑制氧化损伤至关重要，对于使细胞能够利用MN作为抗氧化剂至关重要。发现SOD1 NULL染色设计为高活化的磷酸盐是氧化应力和不可或缺的空气中的。初步结果表明，高细胞质聚磷酸盐（息肉）涉及氧化损伤的严重程度，并且涉及MN和FE的磷酸盐相互作用。我们假设息肉通过隔离Mn和Fe来增强氧化损伤，从而将其可用性限制在Mn-抗氧化剂中，并将其易于氧化损伤易受影响的Fe/S蛋白。当前建议的目的是检验这一假设并阐明MN抗氧化剂的性质。为了确定息肉在氧化应激中的作用，将设计一系列改变息肉代谢的酵母菌菌株。这些菌株将被利用为息肉的大小，含量和细胞定位的变化，以评估息肉对息肉对各种氧化应激指标以及对MN和FE生物利用度的影响。在SOD1 NULL背景中，PTS菌株可用于确定息肉如何影响Mn抑制氧化损伤和Fe可用性，以修复受损的Fe/S簇。此外，我们将通过使用新开发的endor光谱法在整个细胞中使用新开发的Endor光谱应用，直接监测PTS突变体内部的MN和Fe-Olyp相互作用，这是氧化应激抗性的函数。在Toto中，这些实验将准确揭示多磷酸盐如何影响氧化应激以及Mn和Fe在介导其毒性中的作用。此外，将通过使用高通量遗传筛选来确定MN抗氧化活性所需的基因来确定MN抑制氧化应激的机制。 SOD1零酵母将使用转座子库和突变体进行诱变，并将选择显示Mn拯救氧化损伤的突变体。该屏幕旨在选择参与与Mn抗氧化活性的小分子代谢的基因。总体而言，这些研究应充分了解磷酸盐，MN和FE在细胞氧化应激中的作用以及控制MN抑制氧化损伤的因素。对这种类型的研究是理解的核心，也许是归因于氧化应激的众多人类疾病。