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Proteases, inhibitors and receptors and relationship to disease states.

Dr R. N. Pike, Dr J. C. Whisstock

Proteases are involved in a diverse range of biological processes. Their control by inhibitors is critical to the maintenance of health. Proteases also cleave cell surface receptors leading to activation of second messenger systems and cellular responses such as differentiation and proliferation. Micro-organisms use proteases to alter normal biological mechanisms in the host and thus facilitate pathogenesis.

Thrombosis and cardiovascular disease (Dr R. Pike, Dr N. Quinsey, Dr James Whisstock & Dr S. Bottomley)

The focus is to develop an anti-thrombotic serpin. We are working on the interaction between the coagulation protease, factor Xa and the serpin antithrombin. This involves site-directed mutagenesis of residues in both the serpin and enzyme, which are predicted by molecular modelling to be of importance in the interaction between the molecules. The mutants are then tested for their ability to interact and information gained is fed into the design of an antithrombotic serpin. We are also investigating the molecular mechanism underlying heparin binding by antithrombin. Heparin is the major anticoagulant agent in clinical use today and this effect is mediated through its binding to antithrombin.

Projects:

  1. Determinants of the interaction between factor Xa and antithrombin: design of an antithrombotic serpin.
  2. Molecular mechanism of heparin binding by antithrombin: understanding the conformational change induced in antithrombin by heparin.

Nuclear serpins and their protease targets (Dr J. Whisstock & Dr R. Pike in association with Dr S. Bottomley & Dr P. Bird)

We are working on a novel serpin, MENT, which appears to control nuclear cysteine proteases and the final stage of chromatin condensation to inert heterochromatin. Nuclear cysteine proteinases play crucial roles in controlling levels of transcription factors. We aim to identify the protease target of MENT and elucidate its role in controlling cell proliferation. Furthermore we are currently investigating the chromatin condensing aspect of MENT, a vital step in terminal cell differentiation. Both of these processes are vital to normal cellular function. Disruption of either function has implications for a wide range of diseases.

Projects:

  1. Identification of the nuclear cysteine protease inhibited by MENT
  2. Understanding the conformational transitions associated with MENT-mediated DNA binding and chromatin condensation.

Protease activated receptors in health and disease (Dr R. Pike, A/Prof E. Mackie & Dr P. Bird)

Many cell types carry a seven trans-membrane receptor on their surfaces which is activated by a protease cleavage, resulting in an intracellular signal which has a variety of effects, such as differentiation or proliferation.

Activation of a protease-activated receptor by a protease.
Figure 1: Activation of a protease-activated receptor by a protease.

Projects:

  1. Activation of the receptors in the context of bone resorption (relevant to diseases such as osteoporosis). The PARs are present on bone forming cells and we are investigating the effects of PAR activation by proteases on bone resorption.
  2. Activation of PARs on platelets and other blood cells: activation of the PARs on blood cells causes many effects associated with inflammation. We are investigating the mechanism by which this occurs when neutrophils interact with platelets and release proteases such as cathepsin G.
  3. Structure and function of the PARs: the PARs operate by a unique mechanism. We are carrying out structural studies to determine how the receptor works.

The interaction of protease-adhesins from Porphyromonas gingivalis with host proteins and protease-activated receptors. (Dr. R. Pike, A/Prof. E. Mackie, Dr S. Bottomley & Prof. E. Reynolds [University of Melbourne Dental School]) Porphyromonas gingivalis is the causative agent of gum disease, which is the major cause of tooth loss in the developed world. The bacterium secretes powerful proteases (gingipains) which have adhesins specific for target proteins attached to them. Gingipains have been found to carry out several functions which lead to the symptoms of Periodontal disease. "Knockout" mutants of P. gingivalis which are deficient in terms of the gingipains are significantly less virulent than normal and thus these molecules are promising candidates for the development of therapeutic compounds which will interfere with both proteolysis and adhesion and so prevent the disease.

Projects:

  1. Expression and mutagenesis of adhesins from P. gingivalis. The objective is to determine the site of binding on the adhesin for fibrinogen.
  2. The effect of adhesion inhibitors on proteolysis by the cysteine proteases from P. gingivalis.

Proteases from Dichelobacter nodosus as virulence agents (Dr R. Pike, Prof J. Rood (Microbiology) & Dr J. Whisstock)

Dichelobacter nodosus is the causative agent of sheep foot rot, an important disease of livestock in Australia. The organism produces proteases, which are associated with virulence. In particular, point mutations of a serine protease alter the virulence of the organism markedly. This is a joint project between the Biochemistry & Molecular Biology & Microbiology departments.

Projects:

To induce mutations in the D. nodosus serine protease associated with virulence and carry out in vitro enzyme characterisation and in vivo testing of the effects on genetic mutants of the organism.

Proteases from the liver fluke, Fasciola hepatica, as virulence agents (Dr R. Pike & Dr T. W. Spithill).

Fasciola hepatica or liver fluke is responsible for widespread animal and human disease. We are studying the structure and function of the cysteine proteases from F. hepatica with a view towards developing new drugs to combat this parasite. For further details on the projects available, please see the Molecular Parasitology section of the handbook.