The ultimate aim of our projects is to address the fundamental question of how molecular chaperones modulate protein folding in the cell. To this end, we study the role of molecular chaperones in maintaining protein homeostasis using E. coli and yeast as model systems. We are also mapping what we call the chaperone interaction networks in yeast with the ultimate aim of identifying the rules that govern protein folding processes in the cell. Our group employs a battery of approaches including biochemical, biophysical, proteomics, and bioinformatics tools.

SPECIFIC PROJECTS

1. The role of molecular chaperones and enzyme decarboxylases in the acid stress response of E. coli. In their natural habitats, enterobacteria are constantly under assault by a wide array of environmental stresses. One of the most frequently encountered hostile conditions is acid stress. Neutralophiles like Escherichia coli must travel through the host's digestive tract, which includes the stomach and intestine, before reaching the bowel where pathogenesis typically occurs. In the stomach, bacteria must endure a pH of about 2 with an emptying time of approximately two hours before reaching the less acidic environment of the intestinal tract (pH 4 to 5). An organism's ability to withstand acid stress has been directly correlated with its infectious dose. Therefore, bacteria have evolved very complex acid stress response systems. Our understanding of the genetic, biochemical, and biophysical properties of these systems is still very rudimentary. We are studying the mechanisms by which molecular chaperones and enzyme decarboxylases help to maintain protein homeostasis under such conditions. Specific systems that we are currently concentrating on include the ClpXP chaperone-protease system, the RavA/ViaA chaperone system, and the lysine decarboxylase acid stress response enzyme. Our work in this field sheds novel insights into bacterial infectivity.

2. Analysis of Hsp90 cellular functions. Eukaryotic Hsp90 is a ubiquitous molecular chaperone that plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins including steroid hormone receptors and protein kinases. Hsp90 is found to be overexpressed in cancer cells; as a result, Hsp90 is currently a novel anti-cancer drug target. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate Hsp90 activity. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort aimed at elucidating the cellular functions of Hsp90, we had identified two highly conserved novel Hsp90 interactors, termed Tah1 and Pih1 (also called Nop17). Tah1 and Pih1 bind to the chaperone and also associate physically and functionally with the essential AAA+-type helicases Rvb1 and Rvb2 to form what we call the R2TP complex (Rvb1-Rvb2-Tah1-Pih1). The helicases are critical components of several key multiprotein complexes including the chromatin remodeling complexes INO80 and SWR-C, the Tip60 histone acetyltransferase complex, and the telomerase complex. Rvb1 and Rvb2 are also found to be involved in snoRNA maturation and pre-rRNA processing. The R2TP complex is conserved from yeast to humans.

Our recent efforts aimed at analyzing the effect of Tah1 and Pih1 on Hsp90 activity using yeast as model system. This led us to a very surprising finding that suggested a novel role of the chaperone in pre-rRNA processing. We found that Hsp90 and R2TP are involved in the biogenesis of box C/D small nucleolar RNA-protein (snoRNP) complexes that are required for the processing of pre-ribosomal RNA. Our working model is that Hsp90 and R2TP complex function to promote the proper biogenesis of snoRNA-protein complexes, which are subsequently important for pre-rRNA processing and maturation. Our efforts on this project are aimed at elucidating at the molecular level the ultimate effect of Hsp90 and R2TP on ribosome biogenesis. This project sheds further insights into the role of Hsp90, Rvb1, and Rvb2 in cancer.

3. Mapping chaperone interaction networks. Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins or they can lead to the degradation of misfolded and destabilized proteins. They are also known to be involved in many cellular functions, however, a detailed and comprehensive overview of the interactions between chaperones and their cofactors and substrates is still absent. The heat shock proteins Hsp90, Hsp70/Hsp40, and Hsp60/Hsp10 are typical chaperone systems that are highly conserved across organisms. In this project, we are carrying out systematic mapping of the chaperone interaction networks in yeast using a wide range of proteomic and genomic methods. The ultimate goal of the project is to determine the mechanisms that govern protein homeostasis inside the cell.

Alexopoulos, E., Kanjee, U., Snider, J., Houry, W. A., & Pai, E. F. "Crystallization and Preliminary X-ray Analysis of the Inducible Lysine Decarboxylase from Escherichia coli" Acta Crystallographica Section F: Structural Biology and Crystallizatuion Communications 64(8),700-706 (2008).

Gribun, A., Cheung, K. L. Y., Huen, J., Ortega, J., & Houry, W. A. "Yeast Rvb1 and Rvb2 are ATP-Dependent DNA Helicases that Form a Heterohexameric Complex" Journal of Molecular Biology 376(5), 1320-1333 (2008).

Zhao, R., Kakihara, Y., Gribun, A., Huen, J., Yang, G., Khanna, M., Costanzo, M., Brost, R. L., Boone, C., Hughes, T. R., Yip, C. M., & Houry, W. A. "Molecular Chaperone Hsp90 Stabilizes Pih1/Nop17 to Maintain R2TP Complex Activity that Regulates snoRNA Accumulation" Journal of Cell Biology 180(3), 563-578 (2008).

Snider, J., Thibault, G., & Houry, W. A. "The AAA+ superfamily of Functionally Diverse Proteins", Genome Biology 9(4), article 216, 1-8 (2008).

Snider, J. & Houry, W. A. "AAA+ Proteins: Diversity in Function Similarity in Structure", Biochemical Society Transactions 36(1), 72-77 (2008).

Houry, W. A. "Overview of Prokaryotic Molecular Chaperones" in Henry Stewart Talks Series on "Molecular Chaperones: Principles and Diseases", http://www.hstalks.com/molchap/index.htm , Walid A. Houry, Editor (2007).

Yu, A. Y.-H., & Houry, W. A. "ClpP: A Distinctive Family of Cylindrical Energy-Dependent Serine Proteases" FEBS Letters 581(19), 3749-3757 (2007).

Zhao, R., & Houry, W. A. "Molecular Interaction Network of the Hsp90 Chaperone System" in Molecular Aspects of the Stress Response: Chaperones, Membranes, and Networks, Peter Csermely and Laszlo Vigh, eds. (Landes Bioscience) - part of the series Advances in Experimental Medicine and Biology, Vol 594 (2007).

Thibault, G., Yudin, J., Wong, P., Tsitrin, V., Sprangers, R., Zhao, R., & Houry, W. A. "Specificity in Substrate and Cofactor Recognition by the N-Terminal Domain of the Chaperone ClpX" Proceedings of the National Academy of Sciences 103 (47), 17724-17729 (2006).

Thibault, G., Tsitrin, Y., Davidson, T., Gribun, A., & Houry, W. A. "Large Nucleotide-Dependent Movement of the N-Terminal Domain of the ClpX Chaperone" The EMBO Journal 25 (14), 3367-3376 (2006).

Snider, J., & Houry, W. A. "MoxR AAA+ ATPases: A Novel Family of Molecular Chaperones?" Journal of Structural Biology 156 (1), 200-209 (2006).

Snider, J., Gutsche, I. , Lin, M., Baby, S., Cox, B., Butland, G., Greenblatt, J., Emili, A., & Houry, W. A. "Formation of a Distinctive Complex Between the Inducible Bacterial Lysine Decarboxylase and a Novel AAA+ ATPase" Journal of Biological Chemistry 281(3), 1532-1546 (2006).

Wong, K. S. K., & Houry, W. A. "Hsp90 at the Crossroads of Genetics and Epigenetics" Cell Research 16 (9), 742-749 (2006).

Sprangers, R., Gribun, A., Hwang, P. M., Houry, W. A., & Kay , L. E. "Quantitative NMR Spectroscopy of Supramolecular Complexes: Dynamic Side Pores in ClpP are Important for Product Release" Proceedings of the National Academy of Sciences 102(46), 16678-16683 (2005).

Gribun, A., Kimber, M. S., Ching, R., Spranger, R., Fiebig, K. M., & Houry, W. A. "The ClpP Double-Ring Tetradecameric Protease Exhibits Plastic Ring-Ring Interactions and the N-Termini of Its Subunits Form Flexible Loops that are Essential for ClpXP and ClpAP Complex Formation" Journal of Biological Chemistry 280 (16), 16185-16196 (2005).

Zhao, R., & Houry, W. A. "Hsp90: A Chaperone for Protein Folding and Gene Regulation" Biochemistry and Cell Biology 83 (6), 703-710 (2005).

Zhao , R., Davey, M., Hsu , Y.-C., Kaplanek , P., Tong, A., Parsons, A., Cagney, G., Mai, D., Krogan, N., Greenblatt, J., Boone, C., Emili, A., & Houry, W. A. "Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone" Cell 120 (5), 715-727 (2005).