Krabbeborup3274

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site sub-pocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1 catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and non-lysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography-tandem mass spectrometry revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer) and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain. SRI-011381 Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Sulfur is essential for biological processes such as amino acid biogenesis, iron-sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of  their transport mechanism remain poorly characterized. Here, we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein (SBP) FliY binds L-cystine, L-cysteine, and D-cysteine with micromolar affinities. However, binding of the L- and D-enantiomers induced different conformational changes of FliY, where the L- enantiomer/SBP complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo-FliY, more strongly by D-cysteine-bound FliY, and maximally by L-cysteine- or L-cystine-bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independently of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter, and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities yet equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5) is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Since BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes.