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

Abstract

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.

Transcriptional Responses of the Bacterium Burkholderia terrae BS001 to the Fungal Host Lyophyllum sp. Strain Karsten under Soil-Mimicking Conditions

“…S4). In Saccharomyces cerevisiae , such genes are reported to play roles in responses to stress [50]. Furthermore, two other (contiguous) genes, AKAUv1_990014 and AKAUv1_990015 (both predicted to encode conserved proteins of unknown function), were strongly downregulated (log2 fold changes −2.34 and −2.99; Fig.…”

A Novel Small Molecule Methyltransferase Is Important for Virulence in Candida albicans

“…Orf19.633 (hereafter CaCrg1) was annotated as a putative MTase with diagnostic AdoMet-binding motifs 10, 11 . To test if it is a functional MTase, we synthesized a codon-optimized CaCRG1 sequence (Bio Basic Inc) and expressed it via a galactose-inducible promoter from a plasmid in S. cerevisiae (Supplementary Figure 1A).…”

“…Isolation of RNA and hybridization to the microarrays was performed as described 11 . Three independent replicates were used for the analyses.…”

“…Cells were harvested after overnight induction, and CaCrg1 expression was verified by with anti -HA antibodies. Induction and purification of CaCrg1 was performed as described previously 11 .…”

Recent Advances in Developing Insect Natural Products as Potential Modern Day Medicines

“…The anticancer properties of cantharidin result in arrest of the cell cycle in G2/M phase, apoptosis, and oxygen radical damage to DNA [79]. However, the potential of this small molecule and its derivatives in medicine is not confined to their anticancer properties as they have also been reported to have activity against parasites such as Plasmodium falciparum and Leishmania major [80, 81].…”