Pyrimidine salvage enzymes are essential for de novo biosynthesis of Deoxypyrimidine nucleotides in Trypanosoma brucei

© 2016 Leija et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.This work was supported by National Institutes of Health (grants AI078962 and AI034432) to MAP (https://www.niaid.nih.gov) and (grant GM007062) to CL (https://www.nigms.nih. gov), the Welch Foundation (grant I-1257) to MAP and (grant I-1686) to JJK (http://www.welch1.org), and Fundac ̧ão para a Ciência e Tecnologia (FCT, Portugal) SFRH/BD/51286/2010 (http://www.fct.pt) to FRF.info:eu-repo/semantics/publishedVersio

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

ABSTRACT: BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. METHODS: Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. RESULTS: Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. CONCLUSIONS: Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression

A case–control analysis of oral contraceptive use and breast cancer subtypes in the African American Breast Cancer Epidemiology and Risk Consortium

Abstract Introduction Recent oral contraceptive (OC) use has been consistently associated with increased risk of breast cancer, but evidence on specific breast cancer subtypes is sparse. Methods We investigated recency and duration of OC use in relation to molecular subtypes of breast cancer in a pooled analysis of data from the African American Breast Cancer Epidemiology and Risk Consortium. The study included 1,848 women with estrogen receptor-positive (ER+) breast cancer, 1,043 with ER-negative (ER-) breast cancer (including 494 triple negative (TN) tumors, which do not have receptors for estrogen, progesterone, and human epidermal growth factor 2), and 10,044 controls. Multivariable polytomous logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for exposure categories relative to never use, controlling for potential confounding variables. Results OC use within the previous 5 years was associated with increased risk of ER+ (OR 1.46, 95% CI 1.18 to 1.81), ER- (OR 1.57, 95% CI 1.22 to 1.43), and TN (OR 1.78, 95% CI 1.25 to 2.53) breast cancer. The risk declined after cessation of use but was apparent for ER+ cancer for 15 to 19 years after cessation and for ER- breast cancer for an even longer interval after cessation. Long duration of use was also associated with increased risk of each subtype, particularly ER-. Conclusions Our results suggest that OC use, particularly recent use of long duration, is associated with an increased risk of ER+, ER-, and TN breast cancer in African American women. Research into mechanisms that explain these findings, especially the association with ER- breast cancer, is needed

Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains.

Item does not contain fulltextPEG intracellularly! Although long known to enhance residence half-life of peptides in serum and lysates, the effect of PEGylation on biological probes in cells has received only limited attention. Here it is shown that phosphoramidate-linked PEGylated proapoptotic peptides display a dramatically increased stability in Jurkat cell lysate and a homogenous intracellular distribution as well as high apoptotic activity after introduction into cells

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy

Metabolomic analysis of TK c-null cells.

<p>A. Detected pyrimidine and purine bases, nucleosides and nucleotides for cells grown in HMI-19 medium supplemented with normal serum (NS). The ratios (fold change) of metabolite levels in the absence of Tet for 24h compared to cells grown with Tet are plotted. B. HPAEC analysis of nucleotide sugars ±Tet at 24 h. C. Quantitation of dTTP by enzymatic assay ± Tet at 24 and 48 h for cell grown in HMI-19 supplemented with NS. D. Fold change of detected pyrimidine and purine bases, nucleosides and nucleotides ± Tet at 24 h for cells grown in HMI-19 medium supplemented with dialyzed serum (DS). E. Fold change of TCA intermediates ±Tet at 24 h for cells grown in HMI-19 medium supplemented with DS. Metabolites shown for C and D are from the same experiment. Data for additional detected metabolites for the normal serum (A) and dialyzed serum (D and E) studies are presented in Supplemental Figures. All data were collected in biological triplicate and error bars represent the SEM calculated for the ±Tet ratio by Graph Pad Prism using the baseline-correction algorithm. For A, D and E, multiple T test analysis was performed in GraphPad Prism comparing the +Tet and -Tet conditions for each study. Statistical significance was determined without correction for multiple comparisons and without assuming a consistent standard deviation. For C, data were analyzed using one way ANOVA with Dunnett’s multiple comparison test. Metabolites that showed a significant difference between the conditions are marked * P<0.05, ** P<0.01, *** P<0.001. Abbreviations are common nomenclature or have been previously defined except for CP, carbamoyl phosphate, R5P, ribose 5’-phosphate, 7m-guanosine, 7-methyl guanosine, succinate/m-malonic acid, succinate/methyl-malonic acid.</p

<i>T</i>. <i>brucei</i> pyrimidine pathway.

<p>Green lines salvage routes, blue lines <i>de novo</i> pathway, black lines interconversion routes, and the red dotted line indicates a reaction that is not present in trypanosomatids. The numbers above each arrow represent the enzyme catalyzing the reaction (EC number): <b>1–6</b>: carbamoyl phosphate synthase (6.3.5.5), aspartate carbamoyl transferase (2.1.3.2), dihydroorotase (3.5.2.3), dihydroorotate dehydrogenase (1.3.98.1), orotate phosphoribosyltransferase (2.4.2.10), orotidine 5-phosphate decarboxylase (4.1.1.23); <b>7</b> UMP-CMP kinase (2.7.4.14); <b>8</b>: nucleoside diphosphatase (3.6.1.6); <b>9</b>: nucleoside diphosphate kinase (2.7.4.6); <b>10</b>: cytidine triphosphate synthase (6.3.4.2); <b>11</b>: ribonucleoside diphosphate reductase (1.17.4.1); <b>12</b>: thymidylate kinase (2.7.4.9); <b>13</b>: deoxyuridine triphosphatase (dUTPase) (3.6.1.23); <b>14</b>: dihydrofolate reductase-thymidylate synthase (2.1.1.45); <b>15</b>:cytidine deaminase (CDA) (3.5.4.5); <b>16</b>: thymidine kinase (TK)(2.7.1.21); <b>17</b>: uridine phosphorylase (2.4.2.3); <b>18</b>:uracil phosphoribosyltransferase (2.4.2.9); 19: HD-domain 5’-nucleotidase (3.1.3.89); <b>20</b>: UDP-glucose pyrophosphorylase (2.7.7.9); <b>21</b>: UTP N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase (2.7.7.23); <b>22</b>: UDP-glucose 4-epimerase (5.1.3.2). The pathway was constructed based on the annotation described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref010" target="_blank">10</a>] and modified to incorporate results from our studies. Additionally enzyme <b>7</b> was added based on the published report that one of seven encoded adenylate kinases (ADKG) was biochemically characterized and shown to be a UMP-CMP kinase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref016" target="_blank">16</a>].</p

5'-nucleotidase PFAM domain representatives.

<p>5'-nucleotidase PFAM domain representatives.</p