STRUCTURAL GENOMICS OF NOVEL MACROPHAGE PROTEINS ASSOCIATED WITH INFLAMMATORY DISEASE AND CANCER

Pawel Listwan,^a,c Nathan Cowieson,^a Anna Aagaard,^a,c Robert Serek,^a Carmel Walsh,^a, Timothy Ravasi,^a,c Christine Wells,^a,c Thomas Huber,^b David Hume,^a,c Jenny Martin,^a,c and Bostjan Kobe^a,c

^a Department of Biochemistry and Molecular Biology, and Institute for Molecular Bioscience, University of Queensland, St.Lucia QLD 4072, Australia; ^b Department of Mathematics, University of Queensland, St. Lucia QLD 4072; ^cCRC for Chronic Inflammatory Diseases (listwan@uq.edu.au)

Most of the potential pathogens that attempt to invade a mammalian cell fail at the very first stage due to the remarkable effectiveness of innate immunity. The presence of the potential pathogens is detected via receptors that recognise generic non-mammalian structures including cell wall components including lipopolisaccharides, peptidoglycans, lipotechoic acids and microbial DNA [1]. The first line of defense is the macrophage, which comprises 15-20% of the cells in the most organs, and is particularly abundant at the routes of pathogen entry such as lung, skin, gut and genitourinary tract [2]. When the potential pathogen is recognised, the macrophage engulfs and attempts to destroy the foreign organism. The knowledge of regulation of macrophage function will form the basis of two classes of therapeutics. Amplification of the toxic function of macrophages to destroy foreign organisms or tumor cells more effectively is one option, the other being the selective suppression of some components of the macrophage activation response. These then can be used to treat conditions like septicaemia and toxic shock, arthritis, atherosclerosis and other chronic inflammatory diseases the other one.

To define the molecular functions of proteins with roles in macrophages, we set out to characterise them structurally using X-ray crystallography. We identify proteins with roles in macrophages through expression profiling using microarray technology. We select and prioritise the proteins for structural analysis according to a number of criteria such as anticipated insight into function and feasibility for structure determination. We have developed protocols for cloning, expression and crystallography that can be adapted for high-throughput approach. To the best our knowledge, this project is the first structural genomics effort in Australia, and the microarray-to-structure pipeline is unique among the structural genomics initiatives worldwide. Our initial list of 40 proteins resulted in 7 soluble proteins, 2 targets are currently in crystallisation trails and one is ready for MAD data measurement. Currently we are optimising this system and here we present our strategy for the selection, prioritisation, cloning, expression and crystallisation of a number of protein targets.

References

1 Hoffman, J.A., Kafatos, F.C., Janeway, C.A., And Ezekowitz, R.A. (1999) Phylogenetic perspectives in innate immunity, Science 284, 1313-1318.

2 Gordon, S., and Hume, D. A. (1986). Localization and function of tissue macro-phages, Ciba FoundSymp 118, 54-67.