A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Exploring AdoMet-dependent Methyltransferases in Yeast

This work presents the investigation of fungal AdoMet-dependent methyltransferases. The first part of the dissertation focuses on two distinct methyltransferases with previously unknown functions in the budding yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. To characterize these enzymes I used a combinatorial approach that exploits contemporary high-throughput techniques available in yeast (chemical genetics, expression, lipid profiling and genetic interaction analysis) combined with rigorous biological follow-up. First, I showed that S. cerevisiae CRG1 (ScCRG1) is a small molecule methyltransferase that methylates cytotoxic drug cantharidin and is important for maintaining lipid homeostasis and actin cytoskeleton integrity in response to small-molecule cantharidin in the baker’s yeast. Similarly to ScCRG1, orf19.633 in the human fungal pathogen C. albicans (CaCRG1) methylates cantharidin and is important for GlcCer biosynthesis. I also demonstrated that CaCrg1 is a ceramide- and PIP-binding methyltransferase involved in Candida’s morphogenesis, membrane trafficking and fungal virulence. Together, the analysis of two genes in yeast illuminated the important roles of the novel small molecule methyltransferases in coupling drug response to lipid biosynthesis and fungal virulence. In the second part of my dissertation, I present the systematic characterization of the genetic architecture of the yeast methyltransferome by examining fitness of double-deletion methyltransferase mutants in standard and under environmental stress conditions. This analysis allowed me to describe specific properties of the methyltransferome network and to uncover functional relationships among methyltransferases inspiring multiple hypotheses and expanding the current knowledge of this family of enzymes.Ph

A Novel Small Molecule Methyltransferase Is Important for Virulence in <i>Candida albicans</i>

<i>Candida albicans</i> is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of <i>C. albicans</i> pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in <i>C. albicans</i> are small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates, and regulating intra- and interspecies interactions. In this study, we demonstrated that <i>C</i>. <i>albicans</i> Crg1 (CaCrg1) is a <i>bona fide</i> smMTase that interacts with the toxin <i>in vitro</i> and <i>in vivo.</i> We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation, and membrane trafficking. Biochemical and genetic analyses showed that CaCrg1 plays a role in the complex sphingolipid pathway: it binds to exogenous short-chain ceramides <i>in vitro</i> and interacts genetically with genes of glucosylceramide pathway, and the deletion of <i>CaCRG1</i> leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth <i>Galleria mellonella</i>, a model of infection

Mapping the cellular response to small molecules using chemogenomic fitness signatures.

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes