Serious fungal infections are rising due to the increased use of immunosuppressive agents (either employed to sustain organ transplants or as the result of cancer therapy). Membrane sterol and the sterol biosynthetic pathway have long been target sites for antifungal drugs. Two of the most widely used antifungal classes, the polyenes (amphotericin B) target plasma membrane-bound sterols and the azoles (fluconazole, and itraconazole) target the cytochrome P450-mediated step, lanosterol C-14 demethylation. Saccharomyces cerevisiae contains the best characterized sterol pathway at the genetic and molecular levels and is an excellent model system to study the topographical organization of sterol enzymes and the interactions between these enzymes within one or more sterol complexes. The post-squalene pathway leading to ergosterol in fungi or cholesterol in animal cells share eleven enzymatic reactions in addition to a scaffold protein required for efficient C-4 demethylation. In the post-squalene pathway only two enzymes in the cholesterol pathway and three in the ergosterol pathway account for the structural differences between these molecules. One goal of this proposal is to co-immunoprecipitate the sterol biosynthetic enzymes in order to determine whether there is a single enzymatic complex or several interacting complexes leading to end-product sterol. Specific pair wise enzymatic interactions will be evaluated using a membrane two-hybrid system. In addition interactions between sterol biosynthetic enzymes and sterol esterification enzymes will also be studied. A second major goal of this proposal is to investigate how loss of the ERG27 gene product leads to loss of the upstream enzymatic reaction, the oxidosqualene cyclase (Erg7p). This interaction will be studied at the transcriptional and post-translational levels. A number of transcriptional activators and repressors that affect several genes in the sterol pathway will be studied. Among these are the UPC2 and ECM22, YER064c and MOT3. A screen for novel transcriptional regulators of the C-4 demethylation genes will also be undertaken. Finally, we will complete our analysis of the ERG9 promoter which encodes the squalene synthase, the first enzyme in the mevalonate-sterol pathway dedicated to sterol synthesis. ? ?
Showing the most recent 10 out of 28 publications
