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

Chemoselektive Synthese von funktionalen Peptid- und Proteinkonjugaten für intrazelluläre Anwendungen

SUMMARY AND OUTLOOK This work demonstrates the utility of chemoselective reactions for the bioconjugation of pharmaceutically relevant modifications to peptides and proteins for promoting their delivery. Specifically, PEGylation, Lipidation and Cell Penetrating Peptides were conjugated to peptides or proteins in order to (i) increase their stability against proteolysis, (ii) promote their solubility and (iii) facilitate their cellular uptake.ZUSAMMENFASSUNG UND AUSBLICK Diese Arbeit behandelt die Anwendung chemoselektiver Reaktionen auf die Biokonjugation von Peptiden und Proteinen mit pharmazeutisch relevanten Modifikationen. Im Speziellen wurden die PEGylierung, die Lipidierung und das Konjugieren von zellpenetrierenden Peptiden untersucht um Peptide und Proteine (i) gegen Proteolyse zu stabilisieren, (ii) ihre Löslichkeit zu erhöhen und (iii) um ihre zelluläre Aufnahme zu ermöglichen

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells

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

Soluble klotho binds monosialoganglioside to regulate membrane microdomains and growth factor signaling

Abstract: Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho

<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

<i>Hs</i>DCTD rescues the growth defect in <i>Tb</i>TK RNAi and <i>Tb</i>TK null cell lines.

<p>A. Growth curves for <i>TK</i> RNAi cells or <i>TK</i> RNAi cells containing a Tet-regulated expression plasmid for <i>Hs</i>DCTD. Cell growth was monitored ±Tet for the indicated days. Error bars represent SD for triplicate biological replicates. Inset shows a Western blot of <i>Hs</i>DCTD expression ± Tet at 48 h. B. qPCR analysis of <i>Tb</i>TK mRNA expression in both the <i>TK</i> RNAi cell line and the <i>TK</i> RNAi <i>Hs</i>DCTD rescue line (±Tet 48 h). Error bars represent SEM for triplicate data. Data were normalized to the -Tet control, which is in the background of the single allele TK knockout. C. Growth analysis of <i>TK</i> null cells expressing either FLAG-tagged <i>Tb</i>TK (c-null) or FLAG-tagged <i>Hs</i>DCTD under the control of the Tet promoter. Error bars represent SD for triplicate biological replicates. Inset shows western blot analysis of the <i>Hs</i>DCTD <i>TK</i> null cells 2 days and 5 days after Tet withdraw.</p

Deletion of the <i>T</i>. <i>brucei CDA</i> gene induces pyrimidine auxotrophy.

<p>A. Growth curves for <i>CDA</i> null cells grown in HMI-19 or HMI-19 media supplemented with 500 μM dThd. Cell growth was monitored for the indicated days. Error bars represent SD for triplicate biological replicates. B. qPCR analysis of CDA expression in wild-type SM and <i>CDA</i> null cells. Error bars represent SEM for triplicate data. C-E. Growth analysis of <i>CDA</i> null cells supplemented with dThd (C), dUrd (D) or uracil (E) over a range of concentrations 48 h post dThd withdrawl. Error bars represent the range for duplicate biological replicates. dThd and dUrd dose response curves were fitted to the Agonist vs response (three parameters) equation in GraphPad Prism (line represents the fit), to obtain ED₅₀ for growth stimulation. ED₅₀ = 20 μM (2.9–61) for dThd and 6.8 μM (3.8–13) for dUrd, where values in parenthesis represent the 95% confidence interval.</p