The Role of Phosphate Manganese and Iron on Eukaryotic Oxidative Stress

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)在氧化应激保护中的作用是重叠的。虽然SOD1在超氧化物歧化脱毒中的作用机制已经被很好地描述，但对于细胞如何利用锰来抑制氧化损伤而不依赖于SOD酶的了解很少。最近，以酿酒酵母为模型生物，我们报道了适当的磷酸盐代谢对于抑制氧化损伤是重要的，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，超积累磷酸盐的SOD1零菌株是氧化胁迫的，在空气中不能存活。初步结果表明，高胞质多聚磷酸盐(Polyp)是氧化损伤的严重程度的原因，磷酸盐与锰和铁的相互作用也参与其中。我们假设，息肉通过隔离锰和铁来增强氧化损伤，从而限制了它们对锰抗氧化剂和对氧化损伤敏感的必需铁/S蛋白的可用性。当前提议的目的是检验这一假说，并阐明锰抗氧化剂的性质。为了确定息肉在氧化应激中的作用，将对一系列改变息肉新陈代谢的酵母菌株进行改造。这些菌株，以下称为聚磷酸盐可滴定系列(PTS)，将在息肉的大小、含量和细胞定位方面有不同的变化，将被用来评估息肉对各种氧化应激指标以及对锰和铁的生物利用率的影响。在SOD1零背景下，PTS菌株可以用来确定息肉如何影响锰对氧化损伤的抑制以及修复受损的Fe/S簇的铁的有效性。此外，我们将使用新开发的整个细胞的Endor光谱应用程序，直接监测PTS突变体内部作为氧化应激抵抗功能的Mn和Fe-Polyp的相互作用。总之，这些实验将准确地揭示聚磷酸盐如何影响氧化应激，以及锰和铁在调节其毒性中所起的作用。此外，锰抑制氧化应激的机制将通过采用高通量遗传筛选来识别锰抗氧化活性所需的基因来确定。用转座子文库对SOD1零酵母进行诱变，筛选出锰丢失、氧化损伤修复的突变株。这一筛选旨在选择与小分子新陈代谢有关的基因，这些小分子结合并激活锰，以发挥锰的抗氧化活性。总体而言，这些研究应该会对磷酸盐、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提供更好的见解。这类研究是理解和治疗由氧化应激引起的众多人类疾病的核心。 公共卫生相关性：氧自由基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、神经退化和衰老。研究细胞氧化应激抵抗的基本机制对于理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。我们最近已经证明，除了超氧化物歧化酶(SOD)酶外，锰对于维持大气中氧气中的生命至关重要。然而，人们对细胞如何利用锰作为抗氧化剂知之甚少。目前的研究目的是破译细胞内锰的抗氧化活性的机制，特别强调磷酸盐、锰和铁代谢之间的相互作用。

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.