LONDON - Rapid screening systems to identify compounds that selectively inhibit enzymes that carry out protein degradation in cells are likely to become available soon, allowing identification of new drugs to treat conditions such as cancers and diseases that cause muscle wasting.
The development of such screening assays will be made possible by the discovery by researchers in Sweden of a way of detecting activity of the intracellular protein degradation pathway that includes the molecular complex known as the proteasome. They report their work in a paper in the May 2000 Nature Biotechnology titled "Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells."
Maria Masucci, professor of virology at the Karolinska Institute in Stockholm, Sweden, told BioWorld International: "This method could be potentially very useful for identifying new types of substances which inhibit this pathway."
She and her colleagues have submitted a patent application on the technique, and are negotiating with a Swedish biotechnology company on an agreement to develop their work further.
The degradation of proteins inside cells is very closely regulated. Proteins destined for degradation are tagged by small proteins called ubiquitins. Once marked in this way, they become a substrate for the molecular complex called the proteasome, which is a giant protease. The proteasome unfolds the protein, which then enters a channel in the complex where several enzymes are located. These digest the protein, chopping it into small peptides.
"This pathway is essential for cell survival," Masucci said. "The half-life of many different proteins, such as the cyclins which regulate the cell cycle, and pro-apoptotic enzymes, is very carefully controlled. So, if this pathway is altered in some way, many different diseases can result, such as cancers and muscle wasting diseases."
Masucci and her colleagues discovered several years ago, for example, that a protein of the Epstein-Barr virus is able to block the pathway and thus avoid degradation. It has also been shown, she said, that a protein produced by the human papilloma viruses is able to induce degradation of the tumor suppressor protein p53 by the proteasome.
In light of such findings, the team wanted to gain a better understanding of how the ubiquitin-proteasome pathway is regulated, and how it could be modulated. They were hampered, however, by the lack of a simple method of studying the pathway in living cells.
To overcome this problem, they fused the fluorescent marker molecule called green fluorescent protein (GFP) to ubiquitin, with an amino acid in between.
Masucci explained: "If this fusion protein is put into cells, the ubiquitin part will be cleaved off and the first amino acid exposed, thus generating a substrate for the pathway. How efficiently this protein will be degraded depends on the nature of this first amino acid. When the pathway is not very active, the cells will remain fluorescent, but if the pathway is very active, the fluorescent reporter is degraded and the cells will not be fluorescent. This provides a very good technique for measuring the activity of any substance that will modulate the activity of the pathway."
Use of such fluorescent fusion proteins should make it possible to identify different substances that inhibit the pathway at different points. "There is now a great interest in finding different types of drugs that will modulate the pathway," Masucci said. "For example, there are already clinical trials in progress evaluating the use of proteasome inhibitors in the treatment of cancer. If we can block the degradation of pro-apoptotic proteins, cells will die. So if we can work out the correct pharmacological concentration of such substances, one could hope to selectively kill tumor cells."
The channel of the proteasome contains various enzymes, Masucci added, which together generate a particular subset of peptides when breaking down a protein. Inhibiting one or more of these enzymes would give rise to a different family of peptides.
"In this way," Masucci said, "one could imagine changing the antigenicity of cells. Selective modification of the pathway could lead to therapies for autoimmune diseases or allergic diseases, or generally make it possible to modify what cells show to the immune system."
Commenting on the paper in the same issue of Nature Biotechnology, in an article titled "Probing the proteasome pathway," Alfred Goldberg, professor of cell biology at Harvard Medical School in Boston, said the method should prove "very useful." GFP fusions will allow not only automated fluorescent-activated cell sorter analysis, but also high-throughput screening for novel inhibitors, he said.
Goldberg concluded: "It is likely that many investigators will also soon be following the breakdown of proteins of medical importance (such as IkB and p53) by creating similar GFP fusions."