Molecular Microbiology & Immunology

Research Summary:

My laboratory’s long-standing interest in the biosynthetic sorting of membrane-bound and secretory proteins has focused on the structure, function and transcriptional regulation of the molecular chaperones of the endoplasmic reticulum. These proteins are critical components in the folding, assembly and modification of these types of proteins. Currently, we are focusing on two new projects which emerged from these earlier studies: 1) the use of molecular chaperones to produce more effective anti-tumor vaccines and 2) the elucidation of the host response to prion infection.

1. Chaperone-based vaccines (a collaboration with the Wold laboratory). The efficacy of molecular chaperones as adjuvants for the induction of anti-tumor immunity has been demonstrated in many laboratories over the last two decades. Similarly, the exploration of the therapeutic use of poxviruses in cancer treatment has a long and diverse history. These findings have led us to determine whether poxviruses or other vectors would be an efficient and effective way to accomplish the intracellular delivery of molecular chaperones into tumor cells. Once in the cells, the chaperones could sample the peptide epitopes generated within the tumor and, upon lysis of the infected tumor cells, release the chaperones for uptake by the immune system. The chaperones would then provide these tumor-specific peptides to the MHC Class I antigen-presenting molecules for cell-surface expression. In this project, our combined expertise in chaperone structure and function and Vaccinia virus (VV) genetics and viral vector gene therapy will be combined with the experience developed by VirRx, Inc., in murine tumor models. This puts us in an excellent position to test the feasibility of using VV vectors capable of expressing high levels of molecular chaperones to induce therapeutically effective tumor immunity and to further our basic understanding of the role of the chaperones in this process.

2. The host response to prion infection. The need to understand the mechanisms of pathogenesis of the transmissible spongiform encephalopathies (TSE), the causative agent of which is the prion, has acquired an increased urgency in recent years. The recent evidence from the United Kingdom that a new variant of Creutzfeldt-Jakob disease (vCJD) in humans is related to “mad cow” disease, bovine spongiform encephalopathy (BSE), and the observation of an ever-growing outbreak of chronic wasting disease among deer and elk in the United States have brought the issue of emerging prion diseases into the spotlight. These facts, coupled with the realization that these outbreaks were most likely the result of human activity, such as feeding products derived from infected animals to healthy animals, also raises the specter of the use of the prions as agents of bioterrorism. An increased understanding of the way in which prions, unusual “protein only” infectious agents, cause disease may allow us to develop ways to enhance our biological defenses against them. Further, it may enable us to develop reagents and protocols that allow more sensitive detection of the presence of prions in clinical samples and thus allow earlier detection of infection. The general goal of our project is to elucidate both the cellular and molecular response of the host to infection by prions. Currently, in one facet of our study, we are using a tissue culture model system to examine, in detail, early steps in infection and to assess reproducible alterations in cellular physiology that result from prion infection and propagation. We plan to employ microarray analysis and proteomic technologies to probe the molecular response of the cells undergoing prion infection. A second part of our project begins with observation that one of the most intriguing aspects of prion infection is its interaction with the host immune system. Specifically, while the infected host exhibits no specific humoral or cellular immune response against prions, the immune system seems to aid rather than prevent prion propagation. It is likely that infecting prions first come into contact with the innate immune system of the host, specifically resident dendritic cells (DC) of the tissues. This facet of our study is designed to study the interaction between prions and DC and to test the hypothesis that prions evade the immune defenses of the host by inhibiting the differentiation, maturation and/or antigen presenting function of DC.

Publications:

Gunther, R., Srinivasan, M., Haugejorden, S., Green, M., Ehbrecht, I.-M. and Kuntzel, H.
(1993) Functional Replacement of the Saccharomyces cerevisiae Trg1/Pdi1 Protein by Members of the Mammalian Disulfide Isomerase Family. J. Biol. Chem. 268, 7728-7732.

Srinivasan, M., Lenny, N. and Green, M. (1993) Identification of Genomic Sequences That Mediate the Induction of the Endoplasmic Retidulum Stress Protein ERp72 by Protein Traffic. DNA and Cell Biol. 12, 807-822.

Mazzarella, R.A., Marcus, N., Haugejorden, S., Balcarek, J., Baldassare, J.J., Roy, B.,
Li, L.-J., Lee, A.S. and Green, M. (1994) ERp61 is GRP58, a Stress-Inducible Luminal Endoplasmic Reticulum Protein, but is Devoid of Phosphatidylinositide-Specific Phospholipase C Activity. Arch. Biochem. Biophys. 308, 454-460.

Qu, D.-F., Mazzarella, R.A. and Green, M. (1994) Analysis of the Structure, Synthesis of GRP94, an Abundant Stress Protein of the Endoplasmic Reticulum. DNA and Cell Biol. 13, 117-124.

Qu, D.-F., and Green, M. (1995) Folding and Assembly of MHC Class II Molecules in a
Cell-Free System. DNA and Cell Biol. 14, 741-751.

Marcus, N., Shaffer, D., Farrar, P. and Green, M. (1996) Tissue Distribution of Three Members of the Murine Protein Disulfide Isomerase (PDI) Family.Biochim.Biophys.Acta. 1309, (3) 253-260.

Marcus, N. and Green, M. (1997) NF-Y, a CCAAT Box-Binding Protein, is One of the Trans-Acting Factors Necessary for the Response for the Response of the Murine Erp72 Gene to Protein Traffic. DNA and Cell Biol. 16, 1123-1131.

Chen, N.-H., Baudino, T., MacDonald, P.N., Green, M., Kelley, W.M., Burnett, J.W., Buller, M.L. (2000) Selective Inhibition of Nuclear Steroid Receptor Function by a Protein from a Human Tumorigenenic Pox Virus. Virology 274, 17-25.

Haren, M.T., Siddiqui, A.M., Armbrecht, H.J., Kevorkian R.T., Kim, M.J., Haas M.J., Mazza, A., Kumar, V.M., Green, M., Banks, W.A., Morley, J.E. (2010) Gene Expression and Physiologic Profiling of Young Murine Skeletal Muscle During Castration and Testosterone Replacement.. J. Andrology 33, 1-14.

Ikeda, H., Miyatake, M., Koshikawa, N., Ochiai, K., Yamada, K., Kiss, A., Donlin, M.J., Panneton, W.M., Churchill, J.D., Green, M., Siddiqui, A,.M., Leinweber, A.L., Crews, N.R., Ezerskiy, L.A., Rendell, V.R., Belcheva, M.M., Coscia, C.J. (2010) Morphine Modulation of Thrombspondin Levels in Astrocytes and its Implications for Neurite Outgrowth and Synapse Formation. J. Biol Chem. 285, 38415-38427. Epub 2010 Oct 2.