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Regulation of cell death by intracellular serpins

Dr. P. Bird

The importance of proteolysis

Proteases are enzymes that cleave peptide bonds, thereby either degrading target proteins or specifically altering their structure and biological properties. Proteases play pivotal roles in a wide variety of processes ranging from blood coagulation to programmed cell death (apoptosis). For example, the caspases involved in apoptosis are intracellular proteases that cleave and inactivate important house-keeping proteins thus inducing cell death. Because hydrolysis of a peptide bond by a protease is irreversible, it is crucial for the normal functioning of cells and systems that proteases are strictly regulated and that they do not destroy or alter proteins unless signalled to do so. Most proteases are only activated in response to particular signals or situations, but low level or inadvertent activation can have serious consequences. Indeed, it is well known that unregulated proteolysis can destroy normal cells and tissue, and contribute to diseases such as heart attack, stroke, arthritis, emphysema and cancer. 

Regulation of proteases by serpins

Many proteases are controlled by the action of a different class of proteins called serpins . Serpins act as decoy molecules that resemble the true substrates or targets of particular proteases, and once a protease attempts to cleave a serpin it becomes trapped in an irreversible complex that is subsequently removed from the system. Thus serpins act as "protease sinks", removing active enzyme before it can seriously damage surrounding cells or tissue. 

Due to their important role in controlling proteolysis serpin gene families are found in most eukaryotes and prokaryotes. The function and protease targets of many of these serpins are unknown. However most of the work on serpins has been done on the human gene family, as it has been known for some time that serpin deficiency can cause blood clots, faulty complement activation, lung disorders, cancer and dementia. The mechanism of protease inhibition by serpins is well understood, the relationship between serpin mutation and disease has been established, and three-dimensional (crystal) structures of individual serpins and the serpin-protease complex have been solved. Nevertheless, aspects of serpin biology that are not well understood include how serpins are targeted to particular sites in the body, and how a balance between protease and serpin levels is maintained so that the proteolysis occurs only when required.

New intracellular serpins

My group has discovered a new group of human serpins, and has shown that they have counterparts in other species such as mice. The new and distinguishing feature of these serpins is that they are found inside cells, in contrast to most other serpins, which are found on the surface of cells or in the circulation. Some of the cells possessing these new serpins include granulocytes, monocytes, and cytotoxic lymphocytes, which are all cells of the immune system that produce powerful proteases used to destroy microbes, virus-infected or malignant cells. Our working hypothesis is that these serpins are cytoprotective . In other words, they protect individual cells against their own proteases so that they do not self-destruct. For example, we have shown that one of these serpins, PI-9, prevents host cells from undergoing protease-induced apoptosis. In fact it inhibits two distinct types of apoptosis-inducing enzymes =97 granzyme B (a cytotoxin produced by immune cells) and caspases. 

Our investigations into this new group of serpins are aimed at understanding their role(s) within the body. At present we are concentrating on three members of the family, PI-6, PI-8 and PI-9.. To further our understanding we need to know what cells synthesize intracellular serpins, how synthesis is regulated, where they are located within cells, and what proteases they control. Mapping and analysis of their genes enables us to determine whether they are associated with known diseases. In pursuing these studies, we are using up-to-date techniques in biochemistry, and cell and molecular biology, including recombinant protein production, directed mutagenesis, advanced imaging techniques including confocal microscopy, and the generation of "knockout" mice.

Project areas

  1. Interaction of the intracellular serpin PI-9 with pro-apoptotic proteases (Dr. P. Bird, Dr. P. Duggan, Dr. J. Whisstock). PI-9 inhibits granzyme B, a cytotoxic protease produced by CD8+ and natural killer cells. It also inhibits several caspases, including apoptotic and inflammatory members of the family. Using site-directed mutagenesis we have delineated the crucial residues in the PI-9 inhibitory region that are involved in the interactions with granzymes and caspases. In collaboration with researchers in the School of Chemistry, this will enable us to develop small caspase and granzyme inhibitors based on PI-9, potentially for use in the treatment of diseases involving autoimmunity and/or mis-regulated apoptosis.
  2. Cytoprotection by PI-6 and PI-8 (Dr. P. Bird). PI-6 inhibits several serine proteases including trypsin, chymotrypsin and cathepsin G. Recently we have shown that PI-6 also inhibits lysosomal cysteine proteases, and protects cells from death caused by lysosomal disruption. We are investigating the molecular basis for this protective effect of PI-6. PI-8 is thought to inhibit prohormone convertases but its actual target is unknown. We are studying the tissue distribution and likely targets of PI-8.
  3. Nucleocytoplasmic trafficking of intracellular serpins (Dr. P. Bird, Prof D. Jans). We have shown that intracellular serpins shuttle between the nucleus and cytoplasm, and are associated with the surface of specialized secretory lysosomes in particular cell types. We are investigating the signals on the serpins and the trafficking pathways used to establish this pattern of intracellular distribution.
  4. Characterization of serpin knockout mice (Dr. P. Bird, Dr. K. Scarff). We have generated mice lacking the counterpart of PI-6. These mice appear to have defective granulocytes and macrophages. We are investigating their response to infection and stress.
  5. Polymerization of intracellular serpins (Dr. P. Bird, Dr. S. Bottomley). Serpin polymerization is associated with disease, however serpins normally do not polymerize unless mutated or heated at non-physiological temperatures. The intracellular serpins are unusual in that they polymerize readily at physiological temperatures, in the absence of mutation. We are studying the folding and unfolding characteristics of PI-6 and PI-9 to understand the biological significance of this observation. 

Serpin Review

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