My lab focuses on the interactions between lipids (fat molecules) and proteins and how such associations affect cell signaling and are involved in the pathogenesis of human diseases.
Biological membranes are sheets of lipids that form barriers between compartments within cells or, in the case of the plasma membrane, between the cell and its external environment. The types of lipids in a cell membrane can vary and abruptly form small islands of specific types. A perfect example of a unique lipid microenvironment is the neuronal synapse, the place on a neuron where it comes together with another neuron to communicate. Lipids are also linked to many proteins and, once attached, move the protein to specific places in membranes, like synapses.
The enzymatic pathways responsible for adding or removing many types of lipids to and from proteins are known. One notable exception is how palmitate, one type of lipid, is added to a protein. This process is called palmitoylation and, if not functioning properly, results in severe neurological deficits including schizophrenia, severe mental retardation, diabetes and many types of cancer. A family of 23 enzymes (named DHHC1-23) that puts palmitate onto proteins was recently discovered and seven of the members have already been associated with human diseases. Currently the enzyme with the most clear-cut connection to disease is DHHC2. This protein acts as a metastasis suppressor meaning that its proper function is necessary to keep cancer cells from escaping a primary tumor and forming secondary tumors throughout the body. In some cancer cells, the gene that encodes DHHC2 is deleted. When this happens, many types of cancer become much more deadly by metastasizing. To understand the molecular pathway that begins with deletion of DHHC2 and ends in metastasis, we invented a method that allowed us to identify CKAP4 as the protein that would normally be palmitoylated by DHHC2 but is not since DHHC2 is deleted. With this information, we have made several unexpected and remarkable discoveries that have led us to understand some fundamental molecular mechanisms that underlie perhaps all forms of metastasis. We have filed two new patent applications on these processes as we hope they will have a direct impact on the way cancer will be treated in the future. We have also developed very active collaborations with clinical and basic science cancer labs in order to move more quickly and efficiently toward our understanding of the processes and to develop treatments to prevent the metastasis of cancer.
Last year we made the unexpected discovery that palmitoylation of CKAP4 by DHHC2 is a key regulatory step in the pathogenesis of a disease called interstitial cystitis (IC). CKAP4 is the functional, cell-surface receptor for antiproliferative factor (APF) a small sialoglycopeptide secreted by urinary bladder epithelial cells of patients with IC, a debilitating, chronic bladder disorder that affects approximately 1 million people in the United States. APF binds to CKAP4 with very high affinity and profoundly inhibits normal bladder epithelial cell growth resulting in a thinning of the bladder epithelium to one to two cell layers thick. The result of this condition is the formation of ulcers in the bladder, leakage of urine into the abdominal cavity and severe, chronic, debilitating pain. There is currently no effective treatment for IC. In collaboration with Prof. Susan Keay at the University of Maryland, the world's leading expert in the study and treatment of patients with IC, we have shown that if we block palmitoylation of CKAP4, we are able to reverse completely the pathological affects of APF. We have been awarded a provisional patent through the University of Maryland on the development of a treatment for IC based on our discovery. Following up on these discoveries, we have gone on to characterize fundamental mechanisms by which DHHC2-mediated palmitoylation regulates the subcellular distribution of CKAP4 including its translocation to the nucleus of the cell where it regulates the expression of many IC and cancer related genes.
Metastasis is the spread of malignant cells from a primary to secondary tumor and often distant sites in the body and is a major contributor to cancer-related mortality. It is a complex, multistage process in which subsequent stages are dependent on the earlier stages. Many of these steps are regulated by basic cell biological processes that are common to other cell biological processes. The number of genes/proteins that have been validated as suppressors of metastasis is more than 20 by many estimates. Different metastasis suppressors act to disrupt one or more steps in various metastatic signaling cascades thereby keeping primary tumor cells isolated and reducing the likelihood of dying from cancer. DHHC2 is one such metastasis suppressor. Remarkably we discovered that among the 20-plus other metastasis suppressor proteins, more than half are palmitoylated suggesting that palmitoylation, potentially by DHHC2, may be a very high-order regulator of the metastatic process in tumor cells. This project is a collaboration between my lab and the labs of Profs. Dan Welch at University of Alabama, Birmingham and Sonia Planey at the Commonwealth Medical College of Scranton, PA.
Planey, S.L., Keay, S.K., Zhang, C.O. and Zacharias, D.A. (2009) Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling. Mol Biol Cell. 20(5): 1454-63.
Planey, S.L. and Zacharias, D.A. (2009) Identification of protein palmitoylation targets and inhibitors. Expert Opinion on Drug Discovery, DOI 10.1517/17460440903548218.
Planey SL, Zacharias DA. (2009) Palmitoyl acyltransferases, their substrates, and novel assays to connect them (Review). Molecular Membrane Biology; 26: 14-31.
Zhang J, Planey SL, Ceballos C, Stevens SM, Jr., Keay SK, Zacharias DA. (2008) Identification of CKAP4/p63 as a Major Substrate of the Palmitoyl Acyltransferase DHHC2, a Putative Tumor Suppressor, Using a Novel Proteomics Method. Mol Cell Proteomics; 7: 1378-88.
Moran, Timothy J., Casey Laris, Emma Palfreyman, Michael Theileking, Ivana Mikic, David Zacharias, Scott Callaway, Jeffrey Price, Edward Hunter (2004) NFB Nuclear Translocation Validation and Performance in High Throughput Cell Imaging. Assay and Drug Development Technologies. 3(5):483-500.
Zacharias, David A., and Roger Y. Tsien. Biochemistry and Mutagenesis of the Green Fluorescent Protein. In: Green Fluorescent Protein: Properties, Applications and Protocols. pp 83-121. Eds. Chalfie and Kain.
Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. and Tsien, R.Y. (2003) Genetically targeted chromophore-assisted light inactivation. Nature Biotechnology 21: 1505-1508. View PDF file.
Zacharias, D.A. (2002) Sticky caveas in an otherwise glowing report: oligomerizing fluorescent proteins and their use in cell biology. Science's STKE. www.stke.org/cgi/content/full/OC_sigtrans;2002/131/pe23 . View PDF file.
Campbell, R.E., Oded T., Palmer, A. Steinbach, P. Baird, G. Zacharias, D. and Tsien, R. (2002) A monomeric red fluorescent protein. PNAS 99: 7877-7882. View PDF file.
Zacharias, D.A., Violin, J.D., Newton, A.C. and Tsien, R.Y. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296: 913-916. View PDF file.
Strehler, E.E. and Zacharias, D.A. (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pamps. Physiol. Rev 81: 21-50. View PDF file.
Chan, F.K-M., Siegel, R.M., Zacharias, D., Swofford, R., Holmes, K.L., Tsien, R.Y. and Lenardo, M.J. (2001) Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein. Cytometry 44: 361-368. View PDF file.
Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. and Tsien, R.Y. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 276: 29188-29194. View PDF file.
Baird, G.S., Zacharias, D.A., Gross, L., Hoffman, R., Baldridge, K., and Tsien, R.Y. (2000) Biochemistry, mutagenesis and chromophore of dsRED, a red fluorescent protein from coral. PNAS 97: 11984-11989. View PDF file.
Siegel, R.M., Fredricksen, J.K., Zacharias, D.A., Chan, F. K-M., Johnson, M., Lynch, D. Tsien, R.Y. and Lenardo, M.J. (2000) Fas preassociation is essential for transmembrane signaling and dominant inhibition by pathogenic mutations. Science 288: 2354-2357. View PDF file.