ABSTRACT
Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes' steady-state kinetics. Although structural differences near the active site and along the C-terminal α-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development.
Subject(s)
Enzyme Inhibitors/pharmacology , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/chemistry , Amino Acid Sequence , Animals , Biocatalysis , Cloning, Molecular , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Protein ConformationABSTRACT
A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.
Subject(s)
Gene Regulatory Networks , Genome, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Computational Biology , Gene Duplication , Gene Expression Regulation, Fungal , Genes, Fungal , Genetic Fitness , Metabolic Networks and Pathways , Mutation , Protein Interaction Mapping , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The phosphorylation status of the myocyte enhancer factor 2 (MEF2) transcriptional regulator is a critical determinant of its tissue-specific functions. However, due to the complexity of its phosphorylation pattern in vivo, a systematic inventory of MEF2A phosphorylation sites in mammalian cells has been difficult to obtain. We employed modern affinity purification techniques, combined with mass spectrometry, to identify several novel MEF2 phosphoacceptor sites. These include an evolutionarily conserved KSP motif, which we show is important in regulating the stability and function of MEF2A. Also, an indirect pathway in which a protein kinase casein kinase 2 phosphoacceptor site is phosphorylated by activation of p38 MAPK signaling was documented. Together, these findings identify several novel aspects of MEF2 regulation that may prove important in the control of gene expression in neuronal and muscle cells.
