Browsing by Subject "farnesyltransferase"

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Open Access
Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis.
(mSphere, 2016-03) Esher, Shannon K; Ost, Kyla S; Kozubowski, Lukasz; Yang, Dong-Hoon; Kim, Min Su; Bahn, Yong-Sun; Alspaugh, J Andrew; Nichols, Connie B
Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.
Open Access
Structure-Guided Development of Antifungal Protein Farnesyltransferase Inhibitors and DNA Polymerase Engineering
(2021) Wang, You
Eukaryotic human pathogens present a serious threat to global health, causing hundreds of millions of infections with high death rate each year. Fungi and protozoa are two major classes of eukaryotic pathogens. Fungi Cryptococcus neoformans, Candida albicans, and protozoa Plasmodium falciparum are important pathogens from these classes. Although the therapeutics treating infections caused by these species are available, the options of front-line drugs are limited and the drug resistance is emerging and spreading. Therefore, there is a need for new therapeutics. Protein prenylation catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase) is essential to the survival of Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum. The previous biophysical and biochemical studies of FTase and GGTase from these species illustrate their divergence from the human enzymes, providing opportunities to develop species specific FTase or GGTase inhibitors for treating infectious diseases.In this dissertation, we choose to target FTases from Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum by repurposing and derivatizing the well-studied human FTase inhibitors. We first derivatized human FTase inhibitor L-778,123, leading to a novel compound that shows potent inhibition of Cryptococcus neoformans growth with MIC value of 3 µM. The IC50 of the compound is 130 nM in the presence of physiological concentration of phosphate. Crystal structures of the compound bound to Cryptococcus neoformans FTase (CnFTase) shows a distinct binding mode from the starting compound, explaining the inhibition mechanism. Additionally, the compound does not exhibit significant mammalian cell toxicity up to 200 µM in cell based assays. We also derivatized and evaluated another human FTase inhibitor Tipifarnib. The derivatives showed the improved antifungal activity against Cryptococcus neoformans and Candida albicans. Finally, we have developed a new system to produce Plasmodium falciparum FTase for future inhibitor development. The data present in this dissertation could advance the future development of novel treatment for infections caused by eukaryotic human pathogens. Additionally, we report two protein engineering studies. The first addresses stability and overexpression of the telomerase riboprotein complex. Here we engineered the catalytic core complex and the RNA binding domain, and evaluated the capability of using these materials for inhibitor development. In the second study, an intein was inserted into DNA polymerases to produce temperature controlled enzymes. The intein controlled DNA polymerases only showed activities after intein splicing triggered by high temperature (>60oC), enabling the capability of conducting “hot-start” reactions by themselves. We demonstrated that using intein controlled DNA polymerases could reduce the nonspecific amplifications in PCR reactions.