Mitochondrial SOD & Breast Cancer Risk-Mechanism

描述(申请人提供)Val-9Ala多态影响线粒体锰超氧化物歧化酶(MnSOD)输入序列的二级结构。Val等位基因编码的蛋白质在分离的大鼠肝脏线粒体中的输入减少。此外，在日本患者中，Val纯合子增加了非家族性心肌病的风险。相比之下，在绝经前女性中，Ala/Ala基因与乳腺癌风险增加相关。该项目的目标是开发关于这两个MnSOD对线粒体和细胞内活性氧物种(ROS)水平的影响的初步结果，这将导致未来深入研究Ala/Ala基因可能如何导致乳腺癌风险增加。本项目的具体目标是：1)确定线粒体MnSOD蛋白水平、MnSOD活性与超氧阴离子和过氧化氢产生之间的关系。将从几个具有不同MnSOD基因的人乳腺细胞系中分离出线粒体。通过Western blotting测定酶活性和蛋白质水平来确定MnSOD的比活性。还将测定线粒体谷胱甘肽过氧化物酶活性和谷胱甘肽/谷胱甘肽转移酶水平。02的消耗、超氧化物和过氧化氢的产生将在支持线粒体呼吸的基础条件下的孵育期间和在诱导氧化应激的治疗时使用化学发光探针来确定。2)确定从人乳腺细胞分离的线粒体中MnSODAla和MnSODVal的线粒体导入是否不同，通过交叉孵育，这种差异完全是由于MnSOD导入序列的差异，而不是线粒体蛋白质导入能力的差异。线粒体将从人类乳腺细胞系中分离出来，纯合和杂合的多态和导入速率以及水平将使用与报告相连的Ala或Val导入结构域组成的融合蛋白来确定。Val和Ala构建物将分别与Val/Val、Val/Ala和Ala/Ala细胞的线粒体孵育，以进行比较。3)确定ALA/ALA、Val/Val和Val/Ala完整乳腺细胞中MnSOD蛋白水平、MnSOD活性与氧化损伤标志物之间的关系。基础水平和诱导水平的线粒体超氧化物歧化，细胞过氧化氢，线粒体DNA氧化损伤，蛋白质氧化损伤(即蛋白质羰基)，以及铁簇蛋白的活性(即乌头酸酶)将被比较，并与MnSOD蛋白水平和活性相关。4)从单个乳腺细胞系出发，建立表达MnSODAla或MnSODVal的细胞亚系，为未来深入研究Ala/Ala基因导致乳腺癌风险增加的潜在机制提供细胞培养模型。根据将在AIMS 1-3中进行的研究结果，将选择一个合适的乳腺细胞系(S)，并采用基因敲除方法来发展所述的亚系。这种方法的优点是它将提供两个遗传背景和生化相同的亚系，除了MnSOD导入序列的差异

DESCRIPTION (provided by applicant)The Val-9Ala polymorphism affects the secondary structure of the import sequence in mitochondrial manganese superoxide dismutase (MnSOD). The protein encoded by the Val allele exhibited decreased import in isolated rat liver mitochondria. Furthermore, Val homozygotes had an increased risk of nonfamilial cardiomyopathy in Japanese patients. In contrast, the Ala/Ala genotype was associated with increased risk of breast cancer in premenopausal women. The goals of this project are to develop preliminary results pertaining to the effects of these two MnSODs on mitochondrial and cellular levels of reactive oxygen species (ROS) that would lead to future in depth studies on how the Ala/Ala genotype may contribute to increased breast cancer risk. The Specific Aims of this project are to: 1) Determine the relationship between mitochondrial MnSOD protein levels, MnSOD activity, and superoxide and hydrogen peroxide production. Mitochondria will be isolated from several human breast cell lines with different MnSOD genotypes. The specific activity of MnSOD will be determined by determining enzyme activity and protein levels by Western blotting. The activity of mitochondrial glutathione peroxidase and GSH/GSSL levels will also be determined. 02 consumption and superoxide and hydrogen peroxide production will be determined using chemiluminescent probes during incubation under basal conditions that support mitochondrial respiration and upon treatment to induce oxidative stress. 2) Determine if the mitochondrial import of MnSOD Ala and MnSOD Val differ in mitochondria isolated from human breast cells and, through cross incubations, that the differences are solely due to the MnSOD import sequence differences as opposed to differences in mitochondria protein import capabilities. Mitochondria will be isolated from human breast cell lines, homozygous and heterozygous for the polymorphisms and import rates and levels determined using fusion proteins comprised of the Ala or Val import domains linked to a reporter. The Val and Ala constructs will each be incubated with mitochondria from Val/Val Val/Ala and Ala/Ala cells for comparison. 3) Determine the relationships among MnSOD protein levels, MnSOD activity, and markers of oxidative damage in Ala/Ala, Val/Val, and Val/Ala intact breast cells. Basal and induced levels of mitochondria superoxide, cellular hydrogen peroxide, mitochondrial DNA oxidative damage, protein oxidative damage (i.e. protein carbonyls), and activities of iron-cluster proteins (i.e. aconitase) will be compared and related to MnSOD protein level and activity. 4) From a single breast cell line, develop cell sublines expressing either MnSOD Ala or MnSOD Val to provide a cell culture model for future in depth investigation of potential mechanisms behind the increased breast cancer risk associated with the Ala/Ala genotype. Based on the results of the studies to be conducted in aims 1-3, an appropriate breast cell line(s) will be selected and a gene knock out approach used to develop the sublines referred to. The advantage of this approach is that it will provide two sublines of identical genetic backgrounds and biochemistry except for the differences in the MnSOD import sequences