Proteases, Serpins and Cytolysins in Cell Development and Death

Professor Phil Bird
Email:    phil.bird@med.monash.edu.au
Tel:         +61 3 9902 9365

A. Granzymes and perforin

Granzymes are proteases produced by cytotoxic lymphocytes of the immune system. Cytotoxic lymphocytes (Fig 1) destroy virus-infected or cancer cells by releasing granzymes, which enter the cytoplasm via the pore-forming cytolysin perforin to trigger apoptosis. Granzyme B activates caspases, and is one of the most lethal proteases known. How perforin facilitates entry of granzyme B into the target cell cytoplasm is presently unclear.

Figure 1. Coloured Scanning Electron Micrograph of a human Natural Killer (NK) cell attacking a cancer cell.

B. Regulation of proteases by serpins

Serpins trap and inactivate proteases (Fig 2). Serpin deficiency in humans can cause blood clots, immune dysfunction, lung disorders, cancer and dementia. A group of serpins found inside cells protect protease-producing or accessory cells against their own proteases, to prevent unwarranted or untimely death. For example, we have shown that the serpin, PI-9, prevents suicide of cytotoxic lymphocytes caused by exposure to their own granzyme B (Fig 3).

Figure 2. Serpins dynamically trap, distort and inactivate proteases.

Figure 3. PI-9 protects cells from misdirected or mislocated granzyme B.

C. Perforin-like molecules in neural function and cancer

Bone Morphogenetic Protein - Retinoid Acid Inducible Neural Specific Proteins (BRINPs) are highly conserved proteins related to perforin but their molecular functions are entirely unknown. They are mainly expressed in the vertebrate nervous system, and BRINP1 is tumour suppressor gene commonly lost in astrocytomas and bladder cancers.

Examples of Project Areas for Honours and PhD Students

We are using advanced techniques in biochemistry, structural biology, and cell and molecular biology to uncover the pathophysiological roles of granzymes and their serpins, perforin, and BRINPs. These include recombinant protein production, directed mutagenesis, RNA interference, bioinformatics, cell culture, imaging (including confocal microscopy), protein crystallography, and the analysis of model organisms such as "knockout" mice and zebrafish. Projects are available in the following areas:

1. Structure and function of granzymes.
It is now thought that granzymes have functions beyond cytotoxicity, e.g. in cytokine signaling and migration. Granzyme inhibitors may therefore be useful as therapeutic anti-inflammatory agents. We produce granzymes as recombinant proteins in yeast or E.coli, and analyze structure and function using molecular approaches and protein crystallography (with Prof J. Whisstock and Dr. N. Borg). Granzyme inhibitors and bioprobes are being developed with Drs M. Scanlon and J. Simpson (Monash Institute of Pharmaceutical Sciences). Substrates are identified using proteomics, and cleavage site preferences and potential inhibitors are identified by phage display, model peptides and bioinformatics. Uptake into cells is monitored by confocal microscopy and FACS analysis. Granzyme roles in vivo are studied using knockout mice.

2. Perforin.
Perforin dysfunction leads to the severe autoimmune disease haemophagocytic lymphohistiocytosis. In collaboration with Prof James Whisstock and Prof J. Trapani (Peter MacCallum Cancer Institute) we are investigating the structure and function of perforin. We are particularly interested in understanding how perforin facilitates entry of granzymes into the cytoplasm of a target cell.

3. Serpins, lysosomes and cell death.
Lysosomes act as "stress sensors", and release of lysosomal proteases into the cytoplasm causes cell death. In cytotoxic lymphocytes, accidental release of granzyme B from granules (which are specialized lysosomes) would cause death if it exceeds PI-9 levels. We are using cells from knockout mice and approaches including single-cell, time-lapse fluorescent imaging to understand how stress induces release of granzyme B from granules, how PI-9 controls this, and the consequences for the immune system of the animal.

4. BRINPs.
(Collaborative project with Prof J. Whisstock and Dr H. Verkade.)
We have shown that BRINP1 is located in the endoplasmic reticulum, and have characterized a full-length BRINP1 orthologue from zebrafish (92% similarity to human). We are beginning to use zebrafish as a model to study the role of BRINP1 in developing and mature neural tissue, and the effect of disrupting its function (in collaboration with Dr H. Verkade, School of Biological Sciences). We are also examining the cellular distribution of other BRINPs and are searching for binding partners in neural cells.