Microarray Profiling of Messenger RNAs Associated with DHFR Knock-down and DHFR2 Knockout in HepG2 cell line
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ABSTRACT: Dihydrofolate reductase activity is essential for the maintenance of One Carbon Metabolism, as it provides the pathway with tetrahydrofolate (the biologically active form of folate). Most of this cellular activity is due to DHFR. Its paralogue DHFR2 was thought to be responsible for mitochondrial dihydrofolate activity based on recombinant versions of the enzyme. However, the function of the endogenous gene/protein has yet to be assessed. This study aimed at investigating the effects of DHFR2 gene loss in HepG2 cells to help identify its function. Furthermore, the expression profile of the DHFR knockdown was interrogated to assess whether DHFR lowered expression has an impact on DHFR2.
Project description:Methotrexate (MTX) is a potent inhibitor of dihydrofolate reductase (DHFR), where is it used as both an antineoplastic and an immunosuppressant. Mechanisms of MTX resistance in cancers include MTX polyglutamylation and upregulation of DHFR. A series of MTX-based PROteolysis TArgeting Chimeras (PROTACs) were designed to selectively degrade human DFHR. These on-target, cell-active PROTACs show proteosome- and E3 ligase-dependent DHFR degradation, selective degradation of DHFR by proteomics, and interpretable structure-activity relationships. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX, indicating these compounds can complement conventional DFHR enzymatic inhibitors as tool compounds. This chemical probe set, composed of the PROTAC (Diruotrexate), its ester pro-drug (Diruotrexate-ester) and negative control analogs (Diruotrexate-IA1, -IA2), should serve as useful tools for studying one-carbon biochemistry.
Project description:Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial and antiprotozoan chemotherapies. Our in silico amino acid sequence and 3D structure analyses revealed the presence of several putative CK2 phosphorylation sites. Indeed, CK2α subunit phosphorylated DHFR in vitro. In order to identify phosphorylation site we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to in vitro phosphorylation by CK2α subunit. The results pointed to serine 168. Mass spectrometry analyses revealed the presence of additional phosphoserine 145. Phosphorylation by CK2α of S145A mutant and lack of phosphorylation of S145A/S168A double mutant may indicate that S145 phosphorylation may occur only when serine 168 is already phosphorylated. The effect of these and other mutations on enzyme catalytic activity was also investigated.
Project description:The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.
Project description:Investigation of whole genome gene expression level changes in Mycobacterium tuberculosis treated with the DHFR inhibitor WR99210, compared to untreated cells. The antimycobacterial properties of WR99210 are further described in Gerum, A., Ulmer, J., Jacobus, D., Jensen, N., Sherman, D., and C. Sibley. 2002. Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase. Antimicrob Agents Chemother 46(11):3362-3369 [PMID:12384337]
Project description:Saccharomyces cerevisiae DFR1, Dihydrofolate reductase involved in tetrahydrofolate biosynthesis; required for respiratory metabolism; mutation is functionally complemented by human DHFR [Source:SGD;Acc:S000005762], is differentially expressed in 3 experiment(s);
Project description:Saccharomyces cerevisiae DFR1, Dihydrofolate reductase involved in tetrahydrofolate biosynthesis; required for respiratory metabolism; mutation is functionally complemented by human DHFR [Source:SGD;Acc:S000005762], is expressed in 2 baseline experiment(s);
Project description:RNA fragmentation and bisulfite conversion were performed. In brief, 200 ng of mRNAs along with 1 ng in vitro transcribed mouse dihydrofolate reductase (Dhfr) spike-in RNA were used. The bisulfite treated samples were desalted with Nanoseq with 3K omega 500/pk centrifugal devices (PALL). Sequencing was performed on an Illumina HiSeq X-Ten sequencing system. The m5C sites were called using meRanCall from meRanTK (FDR < 0.01). The differential m5C sites were detected