Marius Brouwer - US grants

The long-term goal of the proposed research is a better understanding of the role of copper metallothionein (CuMT) in copper metabolism and in metalloenzyme activation. It is hoped that more detailed knowledge of the components involved in copper-metalloprotein biosynthesis may provide insight into the molecular bases of metabolic disorders that arise from errors in copper metabolism and from decreased activities of copper- dependent enzymes, such as Wilson's disease and Menkes' disease. The more immediate goal of the proposed research is to explore the role of CuMT in the molecular events that lead to the insertion of copper into apohemocyanin (apoHc). In vitro studies on the activation of apoHc will be carried out with a specific metallothionein, CuMT-3, that we have recently purified from the hepatopancreas of the American lobster and the blue crab. In the latter organism there is a direct correlation between levels of Cu(I)MT and Cu(I)- Hc during its molt cycle. This provides us with a unique model system for the study of the involvement of MTs in metalloprotein activation and degradation on a cellular and organismal level. We will establish whether the in vitro activation of apoHc by CuMT-3 requires a direct interaction between the two proteins or whether free Cu(I) in solution is involved in the copper transfer process. We will study the molecular events involved in Hc activation, and the effect of the molting hormone, 20 hydroxyecdysone, on these processes, using suspensions of hepatopancreas cells and radiolabeled forms of copper and cysteine. CuMTs can be separated into multiple forms, and preliminary evidence suggests that these different forms may have different biological functions. As a step towards further clarifying this possibility we will determine if the form of MT which sequesters copper as Hc is broken down is different from the MT that acts as a copper donor during Hc synthesis. Our proposed studies will specifically increase our knowledge of the biological function(s) of the different forms of CuMT and are of general importance to a better understanding of the mechanism of copper insertion into apometalloproteins.

DESCRIPTION (Adapted from the applicant's abstract): Copper is an essential but toxic trace metal that has been implicated in oxidative damage to DNA, proteins, and membranes. The long-term goal of the proposed research is to better understand the mechanisms of copper-induced oxidative stress, and the molecular controls that organisms have developed to defend against oxidative damage. The blue crab, Callinectes sapidus, with its highly-active copper metabolism, is proposed as a nonmammalian model. The immediate goal is to establish the usefulness of the model by measureing baseline data on oxidative stress parameters in the blue crab. Recent work has shown that the intracellular antioxidant glutathione (GSH) in the hepatopancreas of marine crustaceans is involved in the metabolism of copper. GSH reduces Cu(II) to Cu(I), which results in the oxidation of GSH. Cu(I) forms a complex with reduced GSH, which may serve to deliver copper for the biosynthesis of the copper-dependent protein hemocyanin.The presence of three copper-inducible metallothionein genes in the hepatopancreas have also been demonstrated. Through its oxidation of GSH and its catalysis of superoxide and hydroxyl radical formation, copper may play a crucial role in the generation of oxidative stress. On the other hand, copper plays an important role in antioxidant defense by virtue of its catalytic role in the dismutation of superoxide by the enzyme Cu,Zn superoxide dismutase. Furthermore, metallothioneins have been implicated in protection against oxidative stress. These data suggest intricate, but as yet unresolved, relations between copper, GSH, metallothionein, and antioxidant enzymes. The objective of this proposal is to further explore and clarify these relationships. The proposed studies will include exposure of blue crabs and their hepatopancreas tissues to copper and the GSH-depleting agent, buthionine sulfoximine, and measuring the effect of these treatments on: (1) intracellular distribution of copper; (2) amounts of intracellular reduced and oxidized GSH; (3) activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and (4) oxidative tissue damage as measured by the degree of lipid peroxidation. These pilot studies are designed to demonstrate the intricate linkages between copper, GSH, metallothionein, antioxidant enzymes and oxidative damage. The data will provide the basis for long-term studies that will investigate the molecular mechanisms that underlie these linkages, and will help to develop a nonmammalian model for the human disease that are caused or exacerbated by copper-mediated oxidative damage.

; 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 ? 1 w y y y ' T Z K ? j ? 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. *** ;

ABSTRACT
Proposal MCB-0080075
PI: Marius Brouwer

Organisms are dependent on trace metals such as iron and copper for various functions. However, when these metals are in excess they may have toxic effects. The goal of this research project is to further our understanding of the molecular strategies used by organisms to convert potentially toxic copper into a biologically useful form, while avoiding its toxic side effects. Many organisms use metal-specific proteins, called metallothioneins, for detoxification of toxic metals, such as cadmium and mercury, and for detoxification of excess essential metals, such as copper. This project uses the blue crab, an organism that has a very active copper metabolism associated with the synthesis and degradation of hemocyanin, a copper-dependent protein used by the animal to carry oxygen from the gills to the tissues. Earlier work has found that crabs have metallothioneins that are controlled by zinc, as in vertebrates, but also have a copper-specific metallothionein whose synthesis is controlled by copper and not by zinc. This research project has three objectives. First, the hypothesis that the copper-specific metallothionein can provide copper that is required for the biosynthesis of hemocyanin will be examined. These studies may reveal a novel biological function for this new member of the metallothionein family. Secondly, since understanding of the function of a protein at the molecular level requires knowledge of its structure, the three-dimensional structure of the metallothionein protein will be determined. These studies will provide the first structure of a copper-metallothionein of a higher organism. Thirdly, the transcriptional control of the copper-metallothionein gene will be studied. This work may result in the first demonstration of a copper-sensing transcription factor in a higher organism, and may provide the foundation upon which further studies on copper sensing and signaling in higher organisms may be based.