The long-term goal of the research proposed is to understand the cytotoxicity of the fungal ribotoxins, restrictocin, mitogillin and alpha- sarcin. These proteins are made by some Aspergillus sp and are very similar to each other in amino acid sequence and biological activity. All three of these proteins are able to enter animal cells and inactivate the ribosomes of those cells by cleaving the ribosomal RNA at a specific conserved site. A combination of cell entry and inactivation of the ribosomes make these proteins cytotoxic. These same properties increase the potential for using these proteins as therapeutics in cancer, AIDS, or transplant rejection therapies. Understanding the mechanism by which the protein can enter the cell and the parts of the protein responsible for the catalytic activity will aid in designing therapeutics able to kill specific cells. The mechanism used to enter animal cells is unknown and will likely be speculative at least until the crystal or NMR solution structure is determined for the protein.
The specific aims of the research proposed here deal with the ribonuclease activity and structure of restrictocin. We have cloned the cDNA and genomic forms of the gene for restrictocin and have demonstrated the production of restrictocin in transformed strains of Aspergillus nidulans and Aspergillus niger (neither one normally produces restrictocin). Using the reported similarity between restrictocin and ribonuclease U2 we are testing the hypothesis that His- 49, Glu-95, Arg-120 and His-136 are present in the active site of restrictocin. A biological screen was devised for the detection of inactive forms of restrictocin using a form of restrictocin with a codon for methionine replacing the leader sequence and the construction placed under the control of the GAL 1 Saccharomyces cerevisiae promoter. Transformants of S. cerevisiae selected with the URA-3 gene are able to grow in the presence of glucose but not galactose, produce a specific fragment of rRNA and produce restrictocin at levels below immunoblot detection when active restrictocin is expressed. The cDNA clone and PCR techniques were used to alter the coding sequence for Glu-95 and His-I 36. Other similar constructions will be made to change His-49 and Arg-120. One specific mutant tested in this system, H136L, grew in the presence of galactose, did not produce the additional RNA band and accumulated detectable levels of restrictocin whereas E95C and E95G reacted as unmutated restrictocin. These mutant proteins will be produced using the developed expression system for A. nidulans and A. niger and we will determine the effects of altering the specific amino acids detailed above on the activity of the proteins. Proton NMR will be used to test the overall conformation of the mutant forms of the protein. The present expression system will be tested for the expression of restrictocin enriched in 15-N and 13-C which is needed for NMR solution structure determinations. Proteins that will aid in obtaining heavy atom derivatives for crystallographic studies will also be produced during these studies.
| Brandhorst, T; Dowd, P F; Kenealy, W R (2001) The effect of fungal ribosome inactivating proteins upon feeding choice in C. freemani, and indications of a mutualistic relationship with A. restrictus. Environmental mycology. Mycopathologia 152:155-8 |
| Yang, X; Moffat, K (1996) Insights into specificity of cleavage and mechanism of cell entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin. Structure 4:837-52 |
| Brandhorst, T; Dowd, P F; Kenealy, W R (1996) The ribosome-inactivating protein restrictocin deters insect feeding on Aspergillus restrictus. Microbiology 142 ( Pt 6):1551-6 |
| Brandhorst, T; Kenealy, W R (1995) Effects of leader sequences upon the heterologous expression of restrictocin in Aspergillus nidulans and Aspergillus niger. Can J Microbiol 41:601-11 |
