The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans

Huang, Chih‐Mao; Chen, Yee‐Chun; Wu-Hsieh, Betty A.; Fang, Jim‐Min; Chang, Zee-Fen

doi:10.1074/jbc.ra118.006798

Cited by 8 publications

(5 citation statements)

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“…He et al demonstrated that the expression of CAD, a key enzyme in de novo synthesis of pyrimidine, plays a critical role in chemotherapy resistance in STAD [ 52 ]. When the pyrimidine biosynthetic pathway was blocked, the sensitivity of the drug, 5-Fu, to cancer cells, changed [ 53 , 54 ]. We used NMF clustering analysis to identify the genes relevant to pyrimidine metabolism.…”

Section: Discussionmentioning

confidence: 99%

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

Zhang,

Yang,

Kuang

et al. 2024

International Journal of Clinical Practice

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Background. Amino acid metabolism, including ATP production, nucleotide synthesis, and redox homeostatic processes, are associated with proliferation and differentiation of tumor cells. This study aimed to identify novel prognostic biomarkers and potential therapeutic targets of amino acid metabolism-related genes for stomach adenocarcinoma (STAD). Methods. RNA sequencing transcriptome data in the TCGA-STAD (training set) and GTEx datasets (validation set) were used. The LIMMA R program enabled the differentially expressed amino acid metabolism-related genes (AAMRGs) to be found. A prognostic risk score model based on clinical phenotypic features was built using LASSO regression and step multi-Cox analyses. Gene set enrichment analysis (GSEA) was used to find potential molecular pathways associated with STAD. Hierarchical cluster analysis was used to evaluate pyrimidine metabolism. Cultured STAD cells assessed the proliferation of STAD and upregulation of GPX3 expression by CCK8 and flow cytometry. Transwell and wound healing assays assessed the impact of GPX3 on invasion and migration of STAD cells. Western blot and qRT-PCR were used to measure changes in pyrimidine metabolism-related markers and active molecules involved in the AMPK/mTOR signaling pathway. Results. Three AAMRGs, DNMT1, F2R, and GPX3, could independently predict the course of STAD. Pyrimidine metabolism appeared to be significantly associated with these by GSEA and clustering analyses. Pyrimidine metabolism was negatively correlated with GPX3. Functional studies using an overexpressed GPX3 plasmid showed an enhanced migration and invasion of STAD cells as well as the expression of genes associated with pyrimidine metabolism and the AMPK/mTOR signaling pathway. By using a CAD siRNA, it was found that that GPX3 affected 5-fluorouracil resistance during de novo synthesis of pyrimidine through the CAD-UMPS signaling axis. Conclusions. GPX3 which regulates the level of pyrimidine metabolism through the AMPK/mTOR pathway was found to be closely associated with STAD. Our findings demonstrate GPX3 is a reliable biomarker for the prognosis of amino acid metabolism and a probable target for STAD therapy.

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Section: Discussionmentioning

confidence: 99%

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

Zhang,

Yang,

Kuang

et al. 2024

International Journal of Clinical Practice

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“…Studies have investigated polyene susceptibility using CRISPR in C. albicans (Min et al, 2018 ; Huang C.-Y. et al, 2019 ) as well as probed the underlying mechanisms of azole susceptibility in C. albicans (Vyas et al, 2015 ; Liu and Myers, 2017a , b ; Chen et al, 2018 ; Shapiro et al, 2018b ; Nishimoto et al, 2019 ), and other Candida species such as C. orthopsilosis (de San Vicente et al, 2019 ; Morio et al, 2019 ), and the highly azole-resistant C. auris (Kim et al, 2019 ; Rybak et al, 2019 , 2020 ). Similar studies, using mutants created with CRISPR-Cas9, have screened for genes involved in echinocandin susceptibility in C. albicans (Lee et al, 2018 ; Lagree et al, 2020 ) and C. glabrata (Hou et al, 2019 ).…”

Section: Applications Of Crispr In Candidamentioning

confidence: 99%

“…CRISPR-mediated deletion of S. cerevisiae CDC8 and CDC43 homologs in C. albicans and C. glabrata , respectively, led to severe fitness defects; in C. albicans , the absence of Cdc8 kinase also sensitized the strain to the polyene amphotericin B and the pyrimidine 5-FUrd, while the lack of GGTase I CDC43 sensitized multiple Candida species to echinocandins (Huang C.-Y. et al, 2019 ; Sun et al, 2020 ). A novel GGTase I inhibitor, L-269289, also demonstrated fungicidal activity against C. glabrata , suggesting that CRISPR-based investigation of essential genes can lead to potential antifungal candidates (Huang C.-Y.…”

Section: Applications Of Crispr In Candidamentioning

confidence: 99%

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CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

et al. 2021

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The Candida genus encompasses a diverse group of ascomycete fungi that have captured the attention of the scientific community, due to both their role in pathogenesis and emerging applications in biotechnology; the development of gene editing tools such as CRISPR, to analyze fungal genetics and perform functional genomic studies in these organisms, is essential to fully understand and exploit this genus, to further advance antifungal drug discovery and industrial value. However, genetic manipulation of Candida species has been met with several distinctive barriers to progress, such as unconventional codon usage in some species, as well as the absence of a complete sexual cycle in its diploid members. Despite these challenges, the last few decades have witnessed an expansion of the Candida genetic toolbox, allowing for diverse genome editing applications that range from introducing a single point mutation to generating large-scale mutant libraries for functional genomic studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is among the most recent of these advancements, bringing unparalleled versatility and precision to genetic manipulation of Candida species. Since its initial applications in Candida albicans, CRISPR-Cas9 platforms are rapidly evolving to permit efficient gene editing in other members of the genus. The technology has proven useful in elucidating the pathogenesis and host-pathogen interactions of medically relevant Candida species, and has led to novel insights on antifungal drug susceptibility and resistance, as well as innovative treatment strategies. CRISPR-Cas9 tools have also been exploited to uncover potential applications of Candida species in industrial contexts. This review is intended to provide a historical overview of genetic approaches used to study the Candida genus and to discuss the state of the art of CRISPR-based genetic manipulation of Candida species, highlighting its contributions to deciphering the biology of this genus, as well as providing perspectives for the future of Candida genetics.

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“…In addition, changes in dynamics may be important to consider in the design of inhibitors that would be useful in cancer chemotherapy. A better understanding of the human enzyme will also assist in the development of agents which are specific for antimalarial 42 and antifungal 43 thymidylate kinases.…”

Section: ■ Conclusionmentioning

confidence: 99%

Stabilization of Active Site Dynamics Leads to Increased Activity with 3′-Azido-3′-deoxythymidine Monophosphate for F105Y Mutant Human Thymidylate Kinase

2020

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Thymidylate kinases are essential enzymes with roles in DNA synthesis and repair and have been the target of drug development for antimalarials, antifungals, HIV treatment, and cancer therapeutics. Human thymidylate kinase (hTMPK) conversion of the anti-HIV prodrug 3′-azido-3′-deoxythymidine (AZT or zidovudine) monophosphate to diphosphate is the ratelimiting step in the activation of AZT. A point mutant (F105Y) has been previously reported with significantly increased activity for the monophosphate form of the drug [3′-azidothymidine-5′monophosphate (AZTMP)]. Using solution nuclear magnetic resonance (NMR) techniques, we show that while the wild-type (WT) and F105Y hTMPK adopt the same structure in solution, significant changes in dynamics may explain their different activities toward TMP and AZTMP. 13 C spin-relaxation measurements show that there is little change in dynamics on the ps to ns time scale. In contrast, methyl 1 H relaxation dispersion shows that AZTMP alters adenosine nucleotide handling in the WT protein but not in the mutant. Additionally, the F105Y mutant has reduced conformational flexibility, leading to an increase in affinity for the product ADP and a slower rate of phosphorylation of TMP. The dynamics at the catalytic center for F105Y bound to AZTMP are tuned to the same frequency as WT bound to TMP, which may explain the mutant's catalytic efficiency toward the prodrug.

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The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans

Abstract: from the Ministry of Science and Technology, Taiwan. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1 and S2, Tables S1-S3, supporting Methods and Refs. 1-4.

Cited by 8 publications

References 36 publications

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

Stabilization of Active Site Dynamics Leads to Increased Activity with 3′-Azido-3′-deoxythymidine Monophosphate for F105Y Mutant Human Thymidylate Kinase

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