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onsdag 5 juli 2023

DNA replikaatiolle tärkeä " T" . TTP, TDP, TMP, Thymidine Pyrimidiinisynteesin entsyymejä: TK1, TK2, TYMS

https://pubmed.ncbi.nlm.nih.gov/17913703/


Mistä  solu saa sitä TTP allastaan? 

Sen syntetisoimisessa on  tietyt edellytykset, joissa vaaditaan essentiellejä vitamiineja kuten B12 ja Foolihappo.   Ymmärrettävää, että  RNA virukset tyytyvät  tymidiinittömään genomiin. 

https://www.jbc.org/article/S0021-9258(20)54611-5/fulltext

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. 
TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. 
 
 In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1–R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication.
 
 In non-proliferating cells p53R2 substitutes for R2. 
 Catabolic enzymes safeguard the size of the dTTP pool:
 thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP.
 Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies.
 
 Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. 
 
In proliferating cells the
, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.
Mammalian cells contain two separate pools of deoxyribonucleotides: a cytosolic-nuclear pool used for the synthesis of nuclear DNA and a mitochondrial pool for mitochondrial (mt)
DNA synthesis. In the former pool dNTPs are produced from ribonucleotides by the de novo pathway, where ribonucleotide reductase and thymidylate synthase are key enzymes, and by phosphorylation of deoxyribonucleosides via the salvage pathway. mt dNTPs derive from the salvage of deoxyribonucleosides catalyzed by mt kinases and from import of deoxyribonucleotides preformed in the cytosol. Although the mt inner membrane is impermeable to nucleotides, cytosolic and mt dNTP pools communicate via specific transporters. mt carriers for deoxyribonucleotides have been cloned in yeasts (,) and biochemically characterized in mammalian mitochondria (,). Furthermore, early observations on the incorporation of precursors into mtDNA () and our own studies on the dynamics of the mt dTTP pool in cultured human cells (,) provide functional evidence for mt import of thymidine nucleotides from the cytoplasm and demonstrate that in cycling cells the main source of mt dTTP is cytoplasmic de novo synthesis.
Nuclear DNA synthesis is restricted to the S phase of the cell cycle. Ribonucleotide reductase is induced at the transition between G1 and S so that de novo synthesis of cytosolic dNTPs starts concurrently with nuclear DNA replication when the request for precursors is high. Also cytosolic thymidine kinase (TK1) is induced in S phase and contributes to the production of dTTP. Thus cytosolic dNTP pools expand in S phase supporting the needs of nuclear DNA polymerases and then strongly decrease at the exit of mitosis as anaphase-specific proteolysis removes R2, the small subunit of ribonucleotide reductase (), and TK1 ().
A second stable R2 subunit of ribonucleotide reductase has been recently discovered and named p53R2, because its expression is regulated by the tumor suppressor p53, a transcription factor involved in the DNA damage response (,). However, p53R2 is present at a low level also in undamaged cycling cells and is the only small subunit of RNR present in quiescent fibroblasts in culture together with R1 (
); the large subunit is needed to make a functional ribonucleotide reductase. Thus it has now emerged that cells outside S phase have the potential to carry out some de novo synthesis of dNTPs. The level of such synthesis is very low compared with that in cycling cells (), in agreement with the small size of cytosolic dNTP pools in resting cells. However, the R1-p53R2 variant of ribonucleotide reductase fulfills an important function in vivo, as demonstrated by the mtDNA depletion arising in the skeletal muscle of patients with inactivating mutations in RRM2B, the gene coding for p53R2, and in several organs of knockout mice ().mtDNA synthesis is not limited to the S phase of the cell cycle but takes place also in differentiated cells () where nuclear DNA replication has stopped. 
The intramitochondrial salvage pathway for dNTP synthesis starts with the phosphorylation of imported deoxyribonucleosides by the two mt deoxyribonucleoside kinases, thymidine kinase 2 (TK2) and deoxyguanosine kinase, two constitutively expressed enzymes encoded by two nuclear genes (,). The two enzymes have substrate specificities that allow for the phosphorylation of all four deoxyribonucleosides required for DNA synthesis. Isotope-flow experiments with radioactive deoxyribonucleosides indicate that the intramitochondrial salvage pathway is constitutively active (,). Indeed, until the discovery of the pathology linked to p53R2 deficiency, it was considered the main provider of dNTPs for mtDNA replication in quiescent and differentiated cells where the cytosolic pool is very low. We have recently shown that, in cultured human fibroblasts, the size of mt dNTP pools also changes with the proliferation state of the cells, remaining within a few percentage points of the cytosolic pools (). The quantitative changes of mt dNTP pools from cycling to quiescent cells are accompanied by a stronger relative decrease of mt dTTP that alters the proportions of the four dNTPs in the mt pool. The specific behavior of thymidine triphosphate underscores the existence of a separate regulation of this precursor and confirms the importance of dTTP for the maintenance of genomic stability. 
Two mt diseases in humans depend on mutations of genes involved in thymidine metabolism that by altering the mt dTTP pool destabilize the mt genome. Mutations of thymidine phosphorylase (TP), a catabolic enzyme that degrades thymidine and deoxyuridine in the cytosol, cause mt neurogastroencephalomyopathy, a multisystemic syndrome characterized by multiple mutations, deletions, and depletion of mtDNA (). The enzyme loss of function leads to an increased mt dTTP pool that reduces the fidelity of mtDNA synthesis. The acute myopathic form of mtDNA depletion syndrome is instead associated with mutations in TK2 and is attributed to muscle-specific shortage of mt dTTP (). A similar tissue-specific mtDNA depletion is caused by deoxyguanosine kinase deficiency, where the target tissues are instead liver and brain (). Thus, although in both cases the genetic defect is present in all somatic cells, the mt phenotype becomes manifest only in selected tissues, suggesting that the unaffected cells can compensate the mt enzyme deficiency by extramitochondrial pathways.
TK2 shares with TK1 the preferred substrates, i.e. thymidine and deoxyuridine, and in addition phosphorylates deoxycytidine, which is however a much better substrate for the cytosolic deoxycytidine kinase (). Although the two thymidine kinases have the same affinity for thymidine at substrate concentrations below 10 μm (Km values of ∼0.5 μm), the Vmax is 20-fold higher for TK1 (). In cycling cells where TK1 is expressed, TK2 accounts only for a small percentage of total TK activity (
,). In non-cycling cells instead, TK2 is the only thymidine kinase and in cultured quiescent fibroblasts its activity is up-regulated (), suggesting that its role becomes more important when dTTP synthesis in the cytosol is turned down. We previously showed that both TK1 and TK2 participate with two related deoxyribonucleotidases, cdN and mdN, in two futile or “substrate” cycles that in the cytosol and mitochondria, respectively, modulate the relative concentrations of thymidine and its monophosphate, dTMP, keeping under control the dTTP pools in the two compartments (,). Both TK2 and mdN are constitutively expressed (,); therefore, the activity of TK2 is curbed by that of the deoxynucleotidase that catalyzes the opposite reaction. With the experiments presented here we explored the role of TK2 in the maintenance of the mt dTTP pool in relation to that of other two players in the metabolic network: ribonucleotide reductase and TP. We wished to find out under which conditions TK2 function is limiting for the synthesis of mt dTTP and outline a possible basis for the tissue-specific phenotype of TK2 deficiency. Our approach was to silence the TK2 expression by RNA interference, in cycling cells expressing TK1 or devoid of TK1 and in quiescent cells where both TK1 and the S-phase-specific ribonucleotide reductase are inactive, and to measure mt dTTP pool size and the incorporation of exogenous thymidine into this pool. In quiescent fibroblasts we dissected the functional interactions of R1-p53R2 ribonucleotide reductase and TP with TK2 by chemically inhibiting the enzymes in TK2-silenced and non-silenced cells. The availability of thymidine in the extracellular compartment was an important factor in the regulation of the small dTTP pool of quiescent cells.

 GENE TK1   Sytosolinen Tymidiinikinaasi , Thymidine Kinase.cytosolic 17q25.3

NCBI Gene Summary for TK1 Gene

  • The protein encoded by this gene is a cytosolic enzyme that catalyzes the addition of a gamma-phosphate group to thymidine. This creates dTMP and is the first step in the biosynthesis of dTTP, which is one component required for DNA replication. The encoded protein, whose levels fluctuate depending on the cell cycle stage, can act as a low activity dimer or a high activity tetramer. High levels of this protein have been used as a biomarker for diagnosing and categorizing many types of cancers. [provided by RefSeq, Oct 2016]

GeneCards Summary for TK1 Gene

TK1 (Thymidine Kinase 1) is a Protein Coding gene. Diseases associated with TK1 include Thanatophoric Dysplasia, Type I and Hypochondroplasia. Among its related pathways are Pyrimidine metabolism and Mitotic G1 phase and G1/S transition. Gene Ontology (GO) annotations related to this gene include identical protein binding and thymidine kinase activity.

UniProtKB/Swiss-Prot Summary for TK1 Gene

Cell-cycle-regulated enzyme of importance in nucleotide metabolism (PubMed:9575153). Catalyzes the first enzymatic step in the salvage pathway converting thymidine into thymidine monophosphate (PubMed:22385435). Transcriptional regulation limits expression to the S phase of the cell cycle and transient expression coincides with the oscillation in the intracellular dTTP concentration (Probable). Also important for the activation of anticancer and antiviral nucleoside analog prodrugs such as 1-b-d-arabinofuranosylcytosine (AraC) and 3c-azido-3c-deoxythymidine (AZT) (PubMed:22385435). ( KITH_HUMAN,P04183 ) 

Protein details for TK1 Gene (UniProtKB/Swiss-Prot)
Protein Symbol:P04183-KITH_HUMAN
Recommended name:Thymidine kinase, cytosolic
Protein attributes for TK1 Gene
Size:234 amino acidsMolecular mass:25469 Da
Quaternary structure:
Homotetramer (PubMed:15611477, 15733844, 17407781, 22385435, 14697231). Tetramerization from dimerization is induced by ATP and increases catalytic efficiency due to a high affinity for thymidine (PubMed:14697231).Tetramerization is inhibited by phosphorylation at Ser-13 (PubMed:14697231).Interacts (via the KEN box) with FZR1 (PubMed:14701726).
Miscellaneous:Two forms have been identified in animal cells, one in cytosol and one in mitochondria.
  • Activity of the cytosolic enzyme is high in proliferating cells and peaks during the S-phase of the cell cycle; it is very low in resting cells.
  •  
    Molecular function for TK1 Gene according to UniProtKB/Swiss-Prot
Function:Cell-cycle-regulated enzyme of importance in nucleotide metabolism (PubMed:9575153).
  • Catalyzes the first enzymatic step in the salvage pathway converting thymidine into thymidine monophosphate (PubMed:22385435).
    Transcriptional regulation limits expression to the S phase of the cell cycle and transient expression coincides with the oscillation in the intracellular dTTP concentration (Probable).
    Also important for the activation of anticancer and antiviral nucleoside analog prodrugs such as 1-b-d-arabinofuranosylcytosine (AraC) and 3c-azido-3c-deoxythymidine (AZT) (PubMed:22385435). KITH_HUMAN,P04183
CatalyticActivity:

https://www.genecards.org/cgi-bin/carddisp.pl?gene=TK2&keywords=TK

TK2 16q21, Thymidine kinase 2, mitochondrial

  • GeneCards Symbol: TK2 2
  • Thymidine Kinase 2 2 3 5
  • Thymidine Kinase 2, Mitochondrial 2 3 4
  • SCA31 2 3 5
  • Mitochondrial Thymidine Kinase 2 3
  • 2'-Deoxyuridine Kinase TK2 3 4
  • Deoxycytidine Kinase TK2 3 4
  • Mt-TK 3 4
  • EC 2.7.1.21 4
  • EC 2.7.1.74 4
  • EC 2.7.1.- 4
  • TK2-EXT 3
  • MTDPS2 3
  • PEOB3 3
  • MTTK 3

  • This gene encodes a deoxyribonucleoside kinase that specifically phosphorylates thymidine, deoxycytidine, and deoxyuridine. The encoded enzyme localizes to the mitochondria and is required for mitochondrial DNA synthesis. Mutations in this gene are associated with a myopathic form of mitochondrial DNA depletion syndrome. Alternate splicing results in multiple transcript variants encoding distinct isoforms, some of which lack transit peptide, so are not localized to mitochondria. [provided by RefSeq, Dec 2012]

GeneCards Summary for TK2 Gene

TK2 (Thymidine Kinase 2) is a Protein Coding gene. Diseases associated with TK2 include Mitochondrial Dna Depletion Syndrome 2 and Progressive External Ophthalmoplegia With Mitochondrial Dna Deletions, Autosomal Recessive 3. Among its related pathways are Pyrimidine metabolism and Pyrimidine metabolism and related diseases. Gene Ontology (GO) annotations related to this gene include nucleoside kinase activity and thymidine kinase activity. An important paralog of this gene is DCK.

UniProtKB/Swiss-Prot Summary for TK2 Gene
Phosphorylates thymidine, deoxycytidine, and deoxyuridine in the mitochondrial matrix (PubMed:9989599, 11687801). In non-replicating cells, where cytosolic dNTP synthesis is down-regulated, mtDNA synthesis depends solely on TK2 and DGUOK (PubMed:9989599). Widely used as target of antiviral and chemotherapeutic agents (PubMed:9989599). ( KITM_HUMAN,O00142 ) 
Function:
  • Phosphorylates thymidine, deoxycytidine, and deoxyuridine in the mitochondrial matrix (PubMed:9989599, 11687801).
    In non-replicating cells, where cytosolic dNTP synthesis is down-regulated, mtDNA synthesis depends solely on TK2 and DGUOK (PubMed:9989599).
    Widely used as target of antiviral and chemotherapeutic agents (PubMed:9989599). KITM_HUMAN,O00142
CatalyticActivity:
 
 
 THYMIDYLATE synthetase TYMS     
 https://www.genecards.org/cgi-bin/carddisp.pl?gene=TYMS&keywords=Thymidine,synthetase

  • Thymidylate synthase catalyzes the methylation of deoxyuridylate (dU)  to deoxythymidylate (dT)  using, 10-methylenetetrahydrofolate (methylene-THF) as a cofactor. This function maintains the dTMP (thymidine-5-prime monophosphate) pool critical for DNA replication and repair. The enzyme has been of interest as a target for cancer chemotherapeutic agents. It is considered to be the primary site of action for 5-fluorouracil, 5-fluoro-2-prime-deoxyuridine, and some folate analogs. Expression of this gene and that of a naturally occurring antisense transcript, mitochondrial enolase superfamily member 1 (GeneID:55556), vary inversely when cell-growth progresses from late-log to plateau phase. Polymorphisms in this gene may be associated with etiology of neoplasia, including breast cancer, and response to chemotherapy. [provided by RefSeq, Aug 2017]

GeneCards Summary for TYMS Gene

TYMS (Thymidylate Synthetase) is a Protein Coding gene. Diseases associated with TYMS include Dyskeratosis Congenita, Digenic and Dyskeratosis Congenita. Among its related pathways are Pyrimidine metabolism and One-carbon metabolism and related pathways. Gene Ontology (GO) annotations related to this gene include protein homodimerization activity and mRNA binding. An important paralog of this gene is DHFR.

Molecular function for TYMS Gene according to UniProtKB/Swiss-Prot
Function:
  • Catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to thymidine 5'-monophosphate (dTMP), using the cosubstrate, 5,10- methylenetetrahydrofolate (CH2H4folate) as a 1-carbon donor and reductant and contributes to the de novo mitochondrial thymidylate biosynthesis pathway. TYSY_HUMAN,P04818
CatalyticActivity:
 ARTICLE: Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth.

Costantino L., Ferrari S et al.
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, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 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, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
 
Lisäartikkeli:
 
 
 
2000 Dec;279(6):H2735-42.
doi: 10.1152/ajpheart.2000.279.6.H2735.Antiproliferative effect of UTP on human arterial and venous smooth muscle cellsP J White  1 , R Kumari, K E Porter, N J London, L L Ng, M R Boarder
DOI: 10.1152/ajpheart.2000.279.6.H2735 Free article
Abstract We have investigated the hypothesis that responses associated with proliferation are regulated by extracellular nucleotides such as ATP and UTP in cultured human vascular smooth muscle cells (VSMC) derived from internal mammary artery (IMA) and saphenous vein (SV). Platelet-derived growth factor (PDGF), ATP, and UTP each generated an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in both IMA- and SV-derived cells in the absence of detectable inositol 1,4,5-trisphosphate production. ATP alone had no effect on [(3)H]thymidine incorporation into DNA, but with a submaximal concentration of PDGF it raised [(3)H]thymidine incorporation in SV- but not IMA-derived cells. UTP alone also was without effect on [(3)H]thymidine incorporation or cell number. However, in both SV- and IMA-derived cells, UTP reduced the PDGF-stimulated [(3)H]thymidine response and PDGF-stimulated cell proliferation. This cannot be explained by an inhibitory effect on the p42/p44 mitogen-activated protein kinase (MAPK) cascade, since this response to PDGF was not attenuated by UTP. We conclude that, in human VSMC of both arterial and venous origin, UTP acts as an anti-proliferative regulator.
 

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