GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.     

The membrane insertase YidC

The membrane insertases YidC–Oxa1–Alb3 provide a simple cellular system that catalyzes the transmembrane topology of newly synthesized membrane proteins. The insertases are composed of a single protein with 5 to 6 transmembrane (TM) helices that contact hydrophobic segments of the substrate proteins. Since YidC also cooperates with the Sec translocase it is widely involved in the assembly of many different membrane proteins including proteins that obtain complex membrane topologies. Homologues found in mitochondria (Oxa1) and thylakoids (Alb3) point to a common evolutionary origin and also demonstrate the general importance of this cellular process. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Translocons on the inner and outer envelopes of chloroplasts share similar evolutionary origin in Arabidopsis thaliana

The process of importing nuclear encoded proteins into chloroplasts is mediated by the T ranslocons on the O uter/I nner Envelope of C hloroplasts (TOC and TIC complex). The ancestor of the TOC complex was formed by pre‐existing proteins from the cyanobacterial ancestor; other proteins recruited from the host cell or cyanobacterial ancestor were subsequently integrated into the complex. However, little is known about the origin of the TIC complex. In this work, the origin of the TIC complex was investigated through one of its channel proteins, AtTic21. Phylogenetic analysis suggested that AtTic21 is conserved in photosynthetic organisms. AtTic21 showed 33% sequence identity to a Synechocystis protein SynTic21. The successful genetic complementation of an AtTic21 knockout mutant by SynTic21 plus the transit peptide coding sequence of AtTic21 suggested that SynTic21 is an ortholog of AtTic21. The sequence and functional conservation between SynTic21 and AtTic21 suggested that the TIC complex shares a similar evolutionary origin to the TOC complex.     

Glycoproteomics: Past, present and future

This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.     

Action of transglucosidase from Aspergillus niger on maltoheptaose and [U-C]maltose

Oligosaccharides synthesized from a mixture of maltoheptaose and [U-¹³C]maltose with transglucosidase [EC 2.4.1.24] from Aspergillus niger were investigated. When the reaction mixture was incubated at 15 °C for 1 h, several types of oligosaccharides with DP (degree of polymerization) 2 to DP8 containing α-d-Glcp-(1→6)-maltoheptaose were detected by liquid chromatography-mass spectrometry (LC-MS) and methylation analysis. Most of these compounds consisted of α-(1→4) linkages in the main chain and α-(1→6) linkages at the non-reducing ends. However, when the reaction mixture was incubated for 96 h, most of these products were converted into oligosaccharides with DP2 to DP5 consisting of only α-(1→6) linkages. These results suggested that A. niger transglucosidase rapidly transferred glucosyl residues to maltooligosaccharides, and gradually hydrolyzed both α-(1→4) linkages and α-(1→6) linkages at the non-reducing end, and transformed these into smaller molecules of mainly α-(1→6) linkages.     

LC-MS analysis of phospholipids and lysophospholipids in human bronchoalveolar lavage fluid

A reversed phase HPLC method was developed for the simultaneous analysis of different phospholipids and lysophospholipids in human bronchoalveolar lavage fluid (BALF). Separation was achieved using a pellicular C8 column at elevated temperatures with an increasing gradient of acetonitrile containing 0.1% formic acid. Detection was carried out by electrospray ionization ion-trap mass spectrometry. Calibration graphs for selected phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine) showed linearity up to 50 ng allowing quantitative determinations. Identification of the individual species within each class was possible with tandem mass spectrometry. Analysis of BALF phospholipids was performed after liquid/liquid extraction with a mixture of chloroform/methanol/acetic acid. Recoveries ranged from 69 to 97% with standard deviations of less than 6%. The limit of detection varied slightly between different classes but was in the range 0.05-0.25 ng total injected amount.     

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.     

LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation

Docosapentaenoic acid (DPA) is a unique fatty acid that exists in two isomeric forms (n-3 and n-6), which differ in their physiological behaviors. DPA can undergo free radical-mediated peroxidation via lipoxygenase (LOX). 15-LOX, one of the LOX isomers, has received much attention in cancer research because of its very different expression level in normal tissues compared to tumors and some bioactive fatty acid metabolites modulating the tumorigenic pathways in cancer. However, the mechanism linking 15-LOX, DPA metabolites, and their bioactivities is still unclear, and the free radicals generated in DPA peroxidation have never been characterized. In this study, we have studied radicals formed from both soybean and human cellular (PC3-15LOS cells) 15-LOX-catalyzed peroxidation of DPAs at various pH's using a combination of LC/ESR/MS with the spin trapping technique. We observed a total of three carbon-centered radicals formed in 15-LOX-DPA (n-3) stemming from its 7-, 17-, and 20-hydroperoxides, whereas only one formed from 17-hydroperoxide in DPA (n-6). A change in the reaction pH from 8.5 (15-LOX enzyme optimum) to 7.4 (physiological) and to 6.5 (tumor, acidic) not only decreased the total radical formation but also altered the preferred site of oxygenation. This pH-dependent alteration of radical formation and oxygenation pattern may have significant implications and provide a basis for our ongoing investigations of LOXs as well as fatty acids in cancer biology.     

Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts

The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20‐I, atTic20‐IV, atTic20‐II and atTic20‐V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17‐22‐23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20‐I/IV (Group 1) and atTic20‐II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT‐PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T‐DNA mutants were identified. Homozygous tic20‐I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20‐I, none was distinguishable from the wild type. The tic20‐I tic20‐II and tic20‐I tic20‐V double mutants were albino, like the corresponding tic20‐I parent. In contrast, tic20‐I tic20‐IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20‐I and atTic20‐IV was confirmed by complementation analysis. Thus, atTic20‐I and atTic20‐IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2 billion (1.2 × 10⁹) years, they are not essential for normal development.     

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C₁₈ fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.