A Ham1p-Dependent Mechanism and Modulation of the Pyrimidine Biosynthetic Pathway Can Both Confer Resistance to 5-Fluorouracil in Yeast

Carlsson, M.; Gustavsson, Marie; Hu, Guo-Zhen; Murén, Eva; Ronne, Hans

doi:10.1371/journal.pone.0052094

Cited by 16 publications

(33 citation statements)

References 39 publications

(40 reference statements)

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“…Carlsson et al () have previously shown that overexpression of the Ham1 gene in yeast protects against the exogenous supply of the non‐canonical, mutagenic pyrimidine nucleoside 5‐fluorouracil (5‐FU). The yeast Ham1 gene can protect against several non‐canonical nucleosides, including 6‐ N ‐hydroxylaminopurine (Kozmin et al , ; Noskov et al , ), 5‐fluorocytosine, and 6‐azauracil (Carlsson et al , ). To investigate whether U/CBSV Ham1 proteins can also provide protection from 5‐FU, resistance assays were performed.…”

Section: Resultsmentioning

confidence: 99%

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Tomlinson

Pablo‐Rodriguez

Bunawan

et al. 2019

Molecular Plant Pathology

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Summary Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep‐sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild‐type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.

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Section: Resultsmentioning

confidence: 99%

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Tomlinson

Pablo‐Rodriguez

Bunawan

et al. 2019

Molecular Plant Pathology

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“…The open reading frame in each deletion strain has been replaced by the KanMX selection cassette [ 29 ]. The plasmids pCPA1, pCPA2, pHMS1, pYJL055w, pHAM1 have been described previously [ 16 ]. Plasmid pYJL055w is referred to as pLOG1 in the present paper in order to make the genetic nomenclature consistent.…”

Section: Methodsmentioning

confidence: 99%

“…We previously carried out a screen in yeast for genes that confer resistance to 5-FU when overexpressed from a high copy number plasmid [ 16 ]. Among the cloned resistance genes we found HAM1 , which encodes inosine triphosphate pyrophosphatase, an enzyme that dephosphorylates non-canonical purine nucleoside triphosphates ITP, dITP and XTP and thus prevents their incorporation into DNA and RNA [ 17 – 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…Among the cloned resistance genes we found HAM1 , which encodes inosine triphosphate pyrophosphatase, an enzyme that dephosphorylates non-canonical purine nucleoside triphosphates ITP, dITP and XTP and thus prevents their incorporation into DNA and RNA [ 17 – 18 ]. This finding suggested that the Ham1 protein may have a broader specificity than originally thought, targeting non-canonical pyrimidines in addition to purines [ 16 ]. It also raised questions about interactions between the purine and pyrimidine metabolic pathways and the role of such interactions in acquired resistance to nucleotide analogues.…”

Section: Introductionmentioning

confidence: 99%

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Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues

Carlsson

Ronne

2018

PLoS ONE

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Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.

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“…In S . cerevisiae , Ura2 is bifunctional carbamoylphosphate synthetase/aspartate transcarbamylase and catalyzes the first two enzymatic steps in the de novo biosynthesis of pyrimidines [ 51 , 52 ], while it is not verified by direct assays and still uncharacterized in C . albicans .…”

Section: Discussionmentioning

confidence: 99%

Synergistic Antifungal Activity of Berberine Derivative B-7b and Fluconazole

Liu

et al. 2015

PLoS ONE

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Our previous study demonstrated berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. We also suggested BBR played a major antifungal role in the synergism of FLC and BBR, while FLC increased intracellular BBR concentrations. Our following systematic structural modification and reconstruction of BBR core identified the novel scaffold of N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(substituted phenyl)acet-amide derivatives 7a-i, including B-7b and B-7d exhibiting remarkable synergistic antifungal activity and low cytotoxicity. Here, the study mainly investigated the synergistic activity of FLC and B-7b and the underlying mechanism. In vitro interaction of FLC and B-7b was investigated against 30 FLC-resistant clinical isolates of C. albicans and non-C. albicans species, including Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The potent synergistic activity of B-7b in combination with FLC against FLC-resistant C. albicans was found through the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed its better synergism with FLC. And as expected, B-7b exhibited much lower cytotoxicity than BBR to human umbilical vein endothelial cells. In contrast to BBR, we found that endogenous ROS augmentation was not involved in the synergism of FLC and B-7b. According to the results from our present comparative proteomic study, it seemed that the disruption of protein folding and processing and the weakening of cells’ self-defensive ability contributed to the synergism of FLC and B-7b. Together, these results suggested novel scaffold BBR derivative B-7b could be a promising synergist in combination with FLC for the treatment of invasive fungal infections.

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A Ham1p-Dependent Mechanism and Modulation of the Pyrimidine Biosynthetic Pathway Can Both Confer Resistance to 5-Fluorouracil in Yeast

Cited by 16 publications

References 39 publications

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues

Synergistic Antifungal Activity of Berberine Derivative B-7b and Fluconazole

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