Browsing by Subject "S-Adenosylmethionine"

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    A chemical method for labeling lysine methyltransferase substrates.
    (Chembiochem : a European journal of chemical biology, 2011-01) Binda, Olivier; Boyce, Michael; Rush, Jason S; Palaniappan, Krishnan K; Bertozzi, Carolyn R; Gozani, Or
    Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.
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    Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions.
    (Biochemistry, 2010-04-20) Augustus, Anne M; Sage, Harvey; Spicer, Leonard D
    We have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing zero to five specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing zero or one metbox. The affinity of MetJ for all of the DNA sequences studied is enhanced by the addition of SAM, the known cofactor for MetJ in the cell. This effect extends to oligos containing zero or one metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by two or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA and the effect of the corepressor SAM on binding to both specific and nonspecific DNA.
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    One-carbon metabolism during the menstrual cycle and pregnancy
    (PLoS Computational Biology, 2021-12) Reed, Michael; Nijhout, Frederik; Kim, Ruby
    Many enzymes in one-carbon metabolism (OCM) are up- or down-regulated by the sex hormones which vary diurnally and throughout the menstrual cycle. During pregnancy, estradiol and progesterone levels increase tremendously to modulate physiological changes in the reproductive system. In this work, we extend and improve an existing mathematical model of hepatic OCM to understand the dynamic metabolic changes that happen during the menstrual cycle and pregnancy due to estradiol variation. In particular, we add the polyamine drain on S-adenosyl methionine and the direct effects of estradiol on the enzymes cystathionine β-synthase (CBS), thymidylate synthase (TS), and dihydrofolate reductase (DHFR). We show that the homocysteine concentration varies inversely with estradiol concentration, discuss the fluctuations in 14 other one-carbon metabolites and velocities throughout the menstrual cycle, and draw comparisons with the literature. We then use the model to study the effects of vitamin B12, vitamin B6, and folate deficiencies and explain why homocysteine is not a good biomarker for vitamin deficiencies. Additionally, we compute homocysteine throughout pregnancy, and compare the results with experimental data. Our mathematical model explains how numerous homeostatic mechanisms in OCM function and provides new insights into how homocysteine and its deleterious effects are influenced by estradiol. The mathematical model can be used by others for further in silico experiments on changes in one-carbon metabolism during the menstrual cycle and pregnancy.