-
Biochimica Et Biophysica Acta Jan 2014Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide... (Review)
Review
Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Topics: Animals; DNA-Binding Proteins; Humans; Ornithine Decarboxylase; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Unfolding; Proteolysis; Saccharomyces cerevisiae Proteins; Thymidylate Synthase; Transcription Factors; Ubiquitin
PubMed: 23684952
DOI: 10.1016/j.bbamcr.2013.05.008 -
Molecular Medicine (Cambridge, Mass.) Aug 2022The active metabolite of 5-Fluorouracil (5FU), used in the treatment of several types of cancer, acts by inhibiting the thymidylate synthase encoded by the TYMS gene,... (Review)
Review
BACKGROUND
The active metabolite of 5-Fluorouracil (5FU), used in the treatment of several types of cancer, acts by inhibiting the thymidylate synthase encoded by the TYMS gene, which catalyzes the rate-limiting step in DNA replication. The major failure of 5FU-based cancer therapy is the development of drug resistance. High levels of TYMS-encoded protein in cancerous tissues are predictive of poor response to 5FU treatment. Expression of TYMS is regulated by various mechanisms, including involving non-coding RNAs, both miRNAs and long non-coding RNAs (lncRNAs).
AIM
To delineate the miRNAs and lncRNAs network regulating the level of TYMS-encoded protein.
MAIN BODY
Several miRNAs targeting TYMS mRNA have been identified in colon cancers, the levels of which can be regulated to varying degrees by lncRNAs. Due to their regulation by the MALAT1 lncRNA, these miRNAs can be divided into three groups: (1) miR-197-3p, miR-203a-3p, miR-375-3p which are downregulated by MALAT1 as confirmed experimentally and the levels of these miRNAs are actually reduced in colon and gastric cancers; (2) miR-140-3p, miR-330-3p that could potentially interact with MALAT1, but not yet supported by experimental results; (3) miR-192-5p, miR-215-5p whose seed sequences do not recognize complementary response elements within MALAT1. Considering the putative MALAT1-miRNAs interaction network, attention is drawn to the potential positive feedback loop causing increased expression of MALAT1 in colon cancer and hepatocellular carcinoma, where YAP1 acts as a transcriptional co-factor which, by binding to the TCF4 transcription factor/ β-catenin complex, may increase the activation of the MALAT1 gene whereas the MALAT1 lncRNA can inhibit miR-375-3p which in turn targets YAP1 mRNA.
CONCLUSION
The network of non-coding RNAs may reduce the sensitivity of cancer cells to 5FU treatment by upregulating the level of thymidylate synthase.
Topics: Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; RNA, Long Noncoding; RNA, Messenger; Thymidylate Synthase
PubMed: 35922756
DOI: 10.1186/s10020-022-00516-2 -
Archives of Biochemistry and Biophysics Oct 2017Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to... (Review)
Review
Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N-N-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity.
Topics: Flavins; Flavoproteins; Methylation; Tetrahydrofolates; Thymidine Monophosphate; Thymidylate Synthase
PubMed: 28821425
DOI: 10.1016/j.abb.2017.08.011 -
Molecular Biology Reports Jul 2022Parasitic diseases have a serious impact on the world in terms of health and economics and are responsible for worldwide mortality and morbidity. The present review... (Review)
Review
Parasitic diseases have a serious impact on the world in terms of health and economics and are responsible for worldwide mortality and morbidity. The present review features the hybrid targeting involving three main enzymes for the treatment of different parasitic diseases. The enzymes Dihydrofolate reductase, thymidylate synthase, and Serine hydroxy methyltransferase play an essential role in the folate pathway. The present review focuses on these enzymes, which can be targeted against several diseases. It shed light on the past, present, and future of these targets, and it can be assessed that these targets can play a significant role against several infectious diseases. For combating viral and protozoal infectious diseases, these targets in combination should be addressed.
Topics: Communicable Diseases; Humans; Methyltransferases; Serine; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase
PubMed: 35253073
DOI: 10.1007/s11033-022-07266-8 -
Clinical and Experimental Pharmacology... Feb 2021Nucleotide metabolism is the driving force of cell proliferation, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in the initial synthesis of nucleotides....
Nucleotide metabolism is the driving force of cell proliferation, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in the initial synthesis of nucleotides. Previous studies reported that TYMS activity significantly affected the proliferation of tumour cells. However, the diagnostic and prognostic significance of TYMS expression in breast cancer remains unclear. Here, we used the Breast Cancer Integrative Platform (BCIP) to investigate the relationship between progression and prognosis of breast cancer with TYMS expression, and then verified the database analysis using immunohistochemical staining. Our results indicated TYMS expression was greater in breast cancer than adjacent normal tissues and greater in triple-negative breast cancer (TNBC) than non-TNBC tissues. TYMS expression also had significant positive correlations with histological grade, tumour size, and ER negativity, and PR negativity. The increased copy number of the TYMS gene appears to be the reason for its upregulation in breast cancer. Breast cancer patients with higher TYMS expression had poorer prognosis. Our data suggest that TYMS has potential use as a diagnostic and prognostic marker for breast cancer patients.
Topics: Biomarkers, Tumor; Humans; Middle Aged; Prognosis; Thymidylate Synthase; Triple Negative Breast Neoplasms
PubMed: 33030246
DOI: 10.1111/1440-1681.13415 -
Molecules (Basel, Switzerland) May 2016In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate... (Review)
Review
In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.
Topics: Anti-Bacterial Agents; Bacteria; Flavins; Humans; Infections; Kinetics; Multienzyme Complexes; Tetrahydrofolate Dehydrogenase; Thymidine Monophosphate; Thymidylate Synthase
PubMed: 27213314
DOI: 10.3390/molecules21050654 -
Molecules (Basel, Switzerland) Jan 2015Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the... (Review)
Review
Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes-dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)-and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.
Topics: Catalysis; Kinetics; Proteins; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase
PubMed: 25591120
DOI: 10.3390/molecules20011192 -
International Journal of Molecular... Feb 2023Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate...
Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.
Topics: Humans; Adenocarcinoma of Lung; Phosphorylation; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase
PubMed: 36769342
DOI: 10.3390/ijms24033023 -
ELife Dec 2022Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS...
Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, , we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
Topics: Female; Animals; Mice; Humans; Binding Sites; Thymidylate Synthase; Fluorouracil; Ovarian Neoplasms; Enzyme Inhibitors
PubMed: 36475542
DOI: 10.7554/eLife.73862 -
Molecules (Basel, Switzerland) Apr 2019In cells, thymidylate synthases provide the only source of 2'-deoxythymidine-5'-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is... (Review)
Review
In cells, thymidylate synthases provide the only source of 2'-deoxythymidine-5'-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is pivotal for cell survival and proliferation. Two main families of thymidylate synthases have been identified in bacteria, folate-dependent thymidylate synthase (TS) and flavin-dependent TS (FDTS). TS and FDTS are highly divergent enzymes, characterized by exclusive catalytic mechanisms, involving different sets of cofactors. TS and FDTS mechanisms of action have been recently revised, providing new perspectives for the development of antibacterial drugs targeting these enzymes. Nonetheless, some catalytic details still remain elusive. For bacterial TSs, half-site reactivity is still an open debate and the recent evidences are somehow controversial. Furthermore, different behaviors have been identified among bacterial TSs, compromising the definition of common mechanisms. Moreover, the redox reaction responsible for the regeneration of reduced flavin in FDTSs is not completely clarified. This review describes the recent advances in the structural and functional characterization of bacterial TSs and FDTSs and the current understanding of their mechanisms of action. Furthermore, the recent progresses in the development of inhibitors targeting TS and FDTS in human pathogenic bacteria are summarized.
Topics: Amino Acid Sequence; Flavins; Humans; Methyltransferases; Molecular Sequence Data; Protein Structure, Secondary; Thymidylate Synthase
PubMed: 31027295
DOI: 10.3390/molecules24081638