CCS

Many FALS-associated mutations of Cu,ZnSOD (SOD1) are now recognized to cause the death of motor neuron cells through a toxic gain-of-function of the enzyme. There are several theories as to the nature of this new function, but many lines of evidence support the possibility that adventitious reactions catalyzed by the copper ion cofactor are central to the progression of the disease. Recent studies have led to the discovery of a family of proteins that play a central role in the physiological pathways that activate SOD, including soluble metal receptors that deliver copper ions to specific enzyme targets within the cell. The Copper Chaperone for Superoxide dismutase (CCS) is responsible for directly inserting the copper ion cofactor into SOD1. Our recent work on the structure, function and chemical mechanism of the yeast CCS/SOD1 system provides a strong foundation to evaluate potential drugs that can control the activity of SOD1 in vivo. Given the reasonable viability of cells and animals that lack SOD1 entirely, a disruption in the process that supplies the ALS mutant enzymes with copper ions could stand as a uniquely promising approach to intervention in the destructive effects of the disease.

Our understanding of the mechanism of CCS metallochaperone action begins with the determination of metal binding capacities. We have developed methods to successfully bind metal ions to many metallochaperone and metalloregulatory proteins. We are working to determine the native metal stoichiometry of CCS for both copper and zinc as well as the metal-ligand environment. Future work in this area includes the measuring the binding strength of metal chaperones relative to their partners in the metal transfer step of the activation mechanism.

The second branch of this research concerns the elucidation of the metal transfer mechanism. Metal transfer mechanisms between individual proteins are not clearly defined. We are studying the role of protein-protein interactions in the activity of CCS and its target protein, SOD1. Size exclusion chromatography, immunodetection and analytical ultracentrifugation are among the techniques that will be used to isolate and analyze these interactions.

Finally we are planning to design and synthesize protein inhibitors that would prevent the activation of FALS mutant SOD1 by CCS. The initial trials will include peptide mimics that would be engineered to prevent the delivery of copper to SOD1 and thereby inactivate it. These studies will eventually be scaled up to include combinatorial methods and both in vitro and in vivo assays of the effectiveness of the inhibitors.

Collaborators

• Professor Valeria Culotta, Johns Hopkins University


• Professor James Penner-Hahn, University of Michigan


• Professor Amy Rosenzweig, Northwestern University

Publications

• Schmidt, P.J., Rae, T.D., Pufahl, R.A., Strain, J., Hamma, T., O'Halloran, T.V., Culotta, V.C. "Multiple Protein Domains Contribute to the Action of the Copper Chaperone for Superoxide Dismutase" J. Biol. Chem. 1999, 247, 23719-23725.


• Portnoy, M.E., Rosenzweig, A.C., Rae, T., Huffman, D.L., O'Halloran, T.V., and Culotta, V.C. "Structure-Function Analyses of the ATX1 Metallochaperone" J. Biol. Chem. 1999, 274, 15041-15045.

Lamb, L.L., Wernimont, A.K., Pufahl, R.A., O'Halloran, T.V., and Rosenzweig, A.C. "Crystal Structure of the Copper Chaperone for Superoxide Dismutase" Nature Structural Biology, 1999, 6, 724-729.
See News and Views "Helping copper find a home" by T. Poulos, p. 709-711 in the same issue.

• Rosenzweig, A.C., Huffman, D.L., Hou, M.Y., Wernimont, A.K., Pufahl, R.P., and O'Halloran, T.V. "Crystal Structure of the Atx1 Metallochaperone Protein at 1.02 A Resolution" Structure, 1999, 7, 605-617.


• Rae, T.D., Schmidt, P.J., Pufahl, R.A., Culotta, V.C., and O'Halloran, T.V. "Undetectable Free Intracellular Copper: the Requirement of a Copper Chaperone for Superoxide Dismutase" Science 1999, 284, 805-808.
  See Perspective "Free Copper Ions in the Cell" by S.J. Lippard, p. 748-749 in the same issue


• Pufahl, R.A. and O'Halloran, T.V. "Mechanisms of Copper Chaperone Proteins" in Metals and Genetics, B. Sarkar, Ed., New York, Plenum Publishing Corporation, 1999.


• Pufahl, R.A. and C.P. Singer, K.L. Peariso, S.-J. Lin, P. Schmidt, V. Cizewski Culotta, J.E. Penner-Hahn, T.V. O'Halloran, "Metal Ion Chaperone Function of the Soluble Cu(I) Receptor, Atx1" Science 1997, 278, 853-856.
  See Perspective "Delivering Copper Inside Yeast and Human Cells" by J.S. Valentine and E.B. Gralla, pgs. 817-818 in the same issue


• Lin, S., Pufahl, R., Dancis, A., O'Halloran, T.V., Culotta, V.C. "A Role for the Saccharomyces cerevisiae ATXI Gene in Copper Trafficking and Iron Transport" JBC 1997, 272, 9215-9220.