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In their wisdom, the creators of an Institute concerned with nuclear technology saw fit to establish a department of radiation biology. At that time, the small number of well characterized proteolytic enzymes provided convenient tools with which to study the effects of radiation on biological materials. Although this general research area never led very far, the discovery, characterization and function of proteolytic enzymes and their inhibitors has formed the basis of a significant programme of research within its successor, the department of biochemistry and molecular biology.

The discovery, at the Institute, of new proteinases and inhibitors in animals and plants has contributed to the current recognition of the great variety of these enzymes, their classification into families, and their importance in health and disease. Only recently, a completely new family of proteinase inhibitors has been discovered in the department. One of the members, equistatin, comes from the sea anemone, while another, on the basis of a quite unexpected discovery, is thought to play an important role in the immune response against foreign proteins.


Evolvement of research areas


MOLECULAR BIOLOGY AND BIOTECHNOLOGY

The ability to isolate and purify proteinases enabled several 3D structures to be determined in collaboration with the Max Planck Institute in Munich. With the advent of recombinant DNA technology, it became possible to study the structure of the genes for proteinases, to make mutant proteins in order to explore their mechanism of action, and to produce enzymes, present in very small amounts in tissues, in quantities sufficient for structural studies. It therefore became appropriate for the Department to acquire its own X-ray crystallographic facility in 1995, and already the structures of cathepsins, alone and in combination with their specific inhibitors, are helping to explain the intricate molecular basis for controlling the potentially destructive activity of proteinases in vivo. Another of the intriguing findings to emerge from this crystallographic structure work concerns evolutionary economy shown in the papain family of proteinases. Those enzymes whose structures have so far been studied employ a common workhorse structure to cleave the peptide bond of their substrate proteins. However, the biologically important specificity for cleavage exhibited by individual members has evolved by subtle changes in the surface topology and, in some, by the addition of structural loops or peptides.

Highly specific monoclonal antibodies have been produced against various proteinases and their inhibitors and these form the basis of diagnostic kits to estimate the levels of these proteins in health and disease, particularly cancers. With larger patient surveys, sufficient data is becoming available to allow significant correlations to be made, making predictions of the state and prognosis of the disease to be made, which is expected to help in treatment. Results are promising for prognosis in metastatic melanoma, mammary gland cancer and lung cancer.

In plants, proteinase inhibitors also play a defensive role against the attack of insects, interfering with their digestive processes. The gene of equistatin was therefore transferred into potato cells, and new transgenic plants produced which show resistance to the attack of Colorado potato beetle larvae. This research is being carried out in co-operation with a Dutch institute. The levels of proteinases and inhibitors are being monitored under stress as part of a programme aimed at understanding mechanisms by which some plants tolerate drought.

Another long-term project in the department has arisen from the interest of the first head of department in animal toxins. These are phospholipase Az (PLA) enzymes that destroy cell membranes in different target tissues. During evolution, some of them have also acquired the ability to block neuro-muscular transmission on the presynaptic side. One of the interesting problems being addressed is the molecular basis of the specificity that different PLAs show - it is now clear that this is not only due to the catalytic activity. Mutant PLAs have been constructed, enabling the part of the molecular surface associated with specificity to be identified. Some of the toxins show important neurotoxic activity and the neuronal receptors to which they bind are being identified and characterized.

Examination of the structure of genes for PLAs has shown that, unlike genes for most proteins, it is the parts that do not code for the mature enzymes that vary least between species. Studies of one of the genetic elements in the latter regions have revealed the unusual fact that they have, on occasion, been transferred 'horizontally' between species, unlike the usual 'vertical' evolutionary transfer.


J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, Telephone: +386 1 477 39 00, Fax: +386 1 251 93 85
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