Copper-Metallothionein and a Novel Superoxide Dismutase in Organisms that Use Copper for Oxygen Transport

;​R o o t E n t y F R 5 C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l 5个54 @F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT代谢生物化学谢莉·a·格雷夫斯谢莉·a·格雷夫斯@ 7\ e = e 7 j j j j j j j j j 1 w y y y ' T Z K[j] j j j j w ~] [j j j j w]小组认识到将蓝蟹作为研究真核生物细胞内铜运输的模型系统的好处。该建议重点突出，表述清晰。这些合作，特别是与伊恩·m·阿米蒂奇博士领导的研究小组的合作，被视为大大加强了该项目。认为目前的研究计划将产生新的、有趣的和重要的铜金属硫蛋白（CuMT）异构体的结构信息。表格7：这是一份优秀的研究计划，在第三次重新提交给项目后，现在正在被推荐资助。PI仔细地利用了审稿人的意见，并准备了一份组织良好、文字清晰、令人信服的提案。专家小组深信，研究蓝蟹在获取适用于高等动物系统的有关铜代谢的重要信息方面具有优势。该提案获得了三个优秀和一个非常好的评价。我很高兴向大家推荐这个无脊椎动物项目。铜是一种必需但有毒的元素。建议阐明这种金属如何在细胞内转化为生物有用的形式，同时避免铜的毒副作用。蓝蟹将被用作模型系统。蓝蟹依赖铜蛋白血青素（Hc）来运输氧气。四种铜配合物作为螃蟹抵御铜毒性的第一道防线：铜-谷胱甘肽（Cu(I)-GSH）和铜-金属硫蛋白（CuMT）家族的三种异构体。cmt - i和cmt - II实际上是相同的。CuMT-III是与众不同的。Cu(I)-GSH可以传递铜来激活apohemocyanin和apoMT。目前尚不清楚cumt是否参与细胞内铜交换。因此，第一个目标是评估假设，即在GSH作为金属交换介质和GSSG作为铜释放剂的情况下，CuMT-I/II和/或CuMT-III可能将铜转移到apoHc。我们的第二个目标是与耶鲁大学的Ian Armitage博士合作，通过二维核磁共振波谱法确定两种不同的CuMT异构体的金属结合特性和三维结构。抗氧化酶，包括普遍存在的铜锌超氧化物歧化酶（CuSnSOD），在高等动物系统中形成抵御铜诱导的氧化细胞损伤的第二道防线。然而，在螃蟹和十足甲壳类动物中一般不可能检测到CuZnSOD。该项目的第三个目标是确定螃蟹的胞质SOD是一种新蛋白，还是CuZnSOD家族中迄今未知的新成员。铜是一种必需但有毒的微量金属，它与DNA、蛋白质和细胞膜的氧化损伤有关。本研究的长期目标是更好地了解细胞内铜运输的控制，以及铜介导的氧化损伤防御的分子机制。研究将以蓝蟹（Callinectes sapidus）为对象进行，蓝蟹的呼吸色素血青素对铜的需求量很高。蓝蟹体内存在三种金属硫蛋白（低分子量金属解毒蛋白）异构体，它们在结构和功能上各不相同。然而，目前尚不清楚cumt是否参与细胞内铜交换过程。为了找出它们是否存在，将用不同的cumt处理无铜血青素，试图恢复正常的血青素功能。其次，利用二维核磁共振（NMR）技术对蓝蟹子囊膜的结构进行分析。研究将与耶鲁大学的Ian Arnitage博士合作进行。除了氧运输外，铜被认为与锌（Zn）一起在特殊的酶中起作用，以保护细胞免受氧的某些毒性作用。这可能发生在一种叫做Cu/Zn超氧化物歧化酶（SOD）的特殊酶中。因此，该项目的第三个目标是确定这种活性是否存在于蓝蟹中，就像在高等动物系统中一样，如果存在，分离和纯化CuZnSOD，试图确定其结构和作用机制。*** ;

; R o o t E n t r y F R 5 C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l 5 5 4 @ F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT METABOLIC BIOCHEMISRY Shelley A. Graves Shelley A. Graves @ 7\ e = e 7 7 j j j j j j j ı 1 w y y y ' T Z K ı j ı j j j j w ~ " j j j j w Metabolic Biochemistry Panel Summary Brouwer, M. MCB 9506050 The panel recognized the benefits of working with the blue crab as a model system for studying intracellular copper trafficking in eukaryotes. The proposal is well focused and clearly articulated. The collaborations, especially with the research group led by Dr. Ian M. Armitage, are seen as significantly strengthening the project. It is felt that the present research program will yield new, interesting, and important structural information on copper-metallothionein (CuMT) isoforms. Form 7: This is an excellent research proposal which is now being recommended for funding after being resubmitted a third time to the program. The PI has made careful use of revi ewers comments and prepared a well-organized, clearly written, compelling proposal. The panel was convinced of the advantages of working with the blue crab to obtain important information about Cu-metabolism applicable to higher animal systems. The proposal received three excellent and one very good rating. I am pleased to recommend this invertebrate project for funding. Brouwer, M. MCB-9506050 Copper is an essential but toxic element. It is proposed to elucidate how this metal is converted intracellularly into a biologically useful form, while avoiding copper s toxic side effects. Blue crabs will be used as a model system. The blue crab is dependent on the copper-protein hemocyanin (Hc) for oxygen transport. Four copper complexes function as a first line of defense against copper toxicity in the crab: copper-glutathione (Cu(I)-GSH), and three isoforms of the copper-metallothionein (CuMT) family. CuMT-I and II are virtually identical. CuMT-III is distinctive. Cu(I)-GSH can deliver copper for the activation of apohemocyanin and apoMT. It is unknown whether CuMTs can participate in intracellular copper exchange. The first objective then is to evaluate the hypothesis that CuMT-I/II and/or CuMT-III may transfer copper to apoHc with GSH acting as a metal-exchange mediator and GSSG as a copper-releasing agent. Our second objective is to determine the metal-binding properties and three-dimensional structure of the two distinct CuMT isoforms by 2 dimensional-NMR spectroscopy, in collaboration with Dr. Ian Armitage at Yale University. Antioxidant enzymes, including the ubiquitous copper-zinc superoxide dismutase (CuSnSOD), in higher animal systems form a second line of defense against copper-induced oxidative cell damage. However, it has not been possible to detect CuZnSOD in crabs and decapod crustacea in general. The third objective of the project is to determine whether the cytosolic SOD in crabs is a novel protein, or a new, hitherto unknown member of the CuZnSOD family. %%% Copp er is an essential but toxic trace metal that has been implicated in oxidative damage to DNA, proteins, and cell membranes. The long-term objective of this study is to gain a better understanding of the control of intracellular trafficking of copper, and of the molecular mechanisms involved in defense against copper-mediated oxidative damage. Studies will be performed with the blue crab, Callinectes sapidus, which has a high requirement for copper in their respiratory pigment, hemocyanin. There appear to be three metallothionein (low-molecular weight metal-detoxifying proteins) (CuMT) isoforms in the blue crab, which are structurally and functionally different from each other. It is unknown, however, whether the CuMTs participate in intracellular copper-exchange processes. To find out if they do, copper-free hemocyanin will be treated with different CuMTs in an attempt to restore normal hemocyanin function. Secondly, two-dimensional nuclear magnetic resonance (NMR) techniques will be used to determine the structure of blue crab CuMTs. Studies will be performed in collaboration with Dr. Ian Arnitage at Yale University. In addition to oxygen transport, it is believed that Cu operates in special enzymes together with zinc (Zn) to protect cells against certain toxic effects of oxygen. This may occur in a special enzyme called Cu/Zn superoxide dismutase (SOD). Consequently, the third objective of this project is to determine if such an activity exists in the blue crab, as in higher animal systems, and, if so, isolate and purify the CuZnSOD in an attempt to determine its structure and mechanism of operation. *** ;