Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis.

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.

Visualizing Rare Watson-Crick-Like Tautomeric and Anionic Mismatches in DNA and RNA

The central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.”

Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.