Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-l-methionine reveals regulation of TPMT*1 and *3C allozymes

Milek, Miha; Šmid, Alenka; Tamm, Riin; Kuželički, Nataša Karas; Metspalu, Andres; Mlinarič-Raščan, Irena

doi:10.1016/j.bcp.2012.01.010

Cited by 20 publications

(18 citation statements)

References 39 publications

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“…Cells incubated in PBS buffer containing SAM or Met have higher TPMT activity compared to cells incubated in PBS only. The higher TPMT activity in SAM and Met treated cells is not a result of increased protein synthesis, instead the altered enzymatic activity is an effect of increased cellular stability of the TPMT protein41. This is in agreement with our thermal stability results, where TPMTwt as well as TPMT p.Y240C and TPMT p.Y240S were stabilized by the addition of SAM.…”

Section: Discussionsupporting

confidence: 91%

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One amino acid makes a difference–Characterization of a new TPMT allele and the influence of SAM on TPMT stability

Helander

Kahlin

et al. 2017

Sci Rep

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Thiopurine induced toxicity is associated with defects in the thiopurine methyltransferase (TPMT) gene. TPMT is a polymorphic enzyme, with most of the single nucleotide polymorphisms (SNPs) causing an amino acid change, altering the enzymatic activity of the TPMT protein. In this study, we characterize a novel patient allele c.719A > C, named TPMT*41, together with the more common variant *3C c.719A > G, resulting in an amino acid shift at tyrosine 240 to serine, p.Y240S and cysteine, p.Y240C respectively. We show that the patient heterozygote for c.719A > C has intermediate enzymatic activity in red blood cells. Furthermore, in vitro studies, using recombinant protein, show that TPMT p.Y240S is less stable than both TPMTwt and TPMT p.Y240C. The addition of SAM increases the stability and, in agreement with Isothermal Titration Calorimetry (ITC) data, higher molar excess of SAM is needed in order to stabilize TPMT p.Y240C and TPMT p.Y240S compared to TPMTwt. Molecular dynamics simulations show that the loss of interactions is most severe for Y240S, which agrees with the thermal stability of the mutations. In conclusion, our study shows that SAM increases the stability of TPMT and that changing only one amino acid can have a dramatic effect on TPMT stability and activity.

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Section: Discussionsupporting

confidence: 91%

“…Although our results show that both TPMTwt and TPMT p.Y240C can be stabilized by SAM, Milek et al . report a more pronounced Met depletion effect on TPMT p.Y240C protein levels41. In addition, our ITC results indicate that both TPMT p.Y240C and TPMT p.Y240S have substantially weaker affinity for SAM compared to TPMTwt.…”

Section: Discussionsupporting

confidence: 53%

One amino acid makes a difference–Characterization of a new TPMT allele and the influence of SAM on TPMT stability

Helander

Kahlin

et al. 2017

Sci Rep

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“…von Stechow and colleagues also reported that cisplatin treatment increased the concentration of the TPMT substrate, S -adenosylmethionine[26]. Combined with a report that SAM stabilizes TPMT[27], these observations are consistent with a putative role for TPMT in the detoxification of cisplatin. Given that increased SAM levels have been shown to exacerbate cisplatin adverse drug reactions[28] in mice, it is possible that TPMT variants, transcriptionally upregulated by cisplatin and destabilized by the *3A mutations, are unable to utilize the SAM co-substrate potentially leading to a two-fold mechanism of enhanced cisplatin cytotoxicity–SAM metabolite accumulation and diminished cisplatin detoxification.…”

Section: Discussionmentioning

confidence: 58%

Pharmacogenetic variants in TPMT alter cellular responses to cisplatin in inner ear cell lines

Bhavsar¹,

Gunaretnam²,

Li³

et al. 2017

PLoS ONE

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Cisplatin is a highly-effective and widely-used chemotherapeutic agent that causes ototoxicity in many patients. Pharmacogenomic studies of key genes controlling drug biotransformation identified variants in thiopurine methyltransferase (TPMT) as predictors of cisplatin-induced ototoxicity, although the mechanistic basis of this interaction has not been reported. Expression constructs of TPMT*3A, *3B and *3C variants were generated and monitored in cultured cells. Cellular TPMT*3A levels were detected at >20-fold lower amounts than the wild type confirming the unstable nature of this variant. The expression of wild type TPMT (TPMT*1) in two murine ear cell lines, HEI-OC1 and UB/OC-1, significantly mitigated their susceptibility to cisplatin toxicity. Cisplatin treatment induced Tlr4 gene expression in HEI-OC1 cells and this response was blunted by the expression of wild type TPMT but not TPMT*3A. In line with the significant mitigation of TPMT*1-expressing cells to cisplatin cytotoxicity, these findings demonstrate a drug-gene interaction between increased TPMT activity and decreased susceptibility to cisplatin-induced toxicity of inner ear cells.

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“…Regardless of its label, the contributions of SAM to the overall stability of TPMT have lately been the focus of a lot of discussion within the field. However, no observations of thermodynamic stabilization of TPMT by SAM binding has (to our knowledge) been published to date, although studies have demonstrated a decrease in the cellular longevity of the protein in cofactor deficient cells [67]. We have therefore investigated properties of SAM binding to TPMT and found that despite exhibiting strong binding to the protein (K D ∼ 2 µm), SAM does not contribute significantly to the stability of TPMT under our experimental conditions.…”

Section: What's the Matter With Sam?mentioning

confidence: 82%

Thiopurine S-methyltransferase - characterization of variants and ligand binding

Blissing¹

2017

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Populärvetenskaplig sammanfattningProteinet TPMT och tiopurinläkemedel I kroppens celler finns proteinet tiopurinmetyltransferas, vilket vanligtvis förkortas TPMT. Trots att det är vanligt förekommande är proteinets naturliga funktion fortfarande okänd. Vi vet dock att TPMT inaktiverar en viss typ av läkemedel som kallas tiopuriner, därav proteinets namn. Tiopuriner är en typ av cytostatika (cellgifter) som använd vid behandling av sjukdomstillstånd relaterade till ett överaktivt immunförsvar och vissa former av leukemi. Tiopurinläkemedel är förstklassiga imitatörer; de efterliknar nämligen byggstenarna till vårt DNA. Cellen kan inte skilja imitatör från original utan bygger in tiopurinerna i nytt DNA. DNA som innehåller tiopuriner är odugligt, vilket gör att cellen till slut dör. Eftersom cellen kopierar sitt DNA före delning så kommer tiopurinläkemedel i större utsträckning påverka celler som delar sig ofta, och man får därmed en riktad effekt mot överaktiva immunceller och cancerceller. TPMT komplicerar läkemedelsbehandlingProteinet TPMT inaktiverar tiopurinläkemedel, och det får tyvärr konsekvenser för patienter som behandlas med dessa substanser. Inaktiveringen sker genom att proteinet modifierar tiopurinerna genom så kallad metylering. De modifierade läkemedelsmolekylerna liknar inte längre de naturliga DNA-baserna, och läkemedlets celldödande effekt minskar därmed. För att kompensera för TPMTs härjningar måste läkemedelsdosen höjas så att tillräcklig effekt uppnås. Dock finns det flertalet naturligt förekommande varianter av proteinet, vilket ytterligare komplicerar behandlingen. Att det finns olika varianter av ett protein är en naturlig i del av den genetiska variationen i en population, men alla TPMTvarianterna inte är lika benägna att inaktivera tiopurinläkemedel. Vissa varianter är ambitiösa och inaktiverar en större mängd tiopurinerde är alltså högaktiva. Andra är betydligt sämre på att inaktivera läkemedelsmolekyler -de är med andra ord lågaktiva. En konsekvens av varianternas olika egenskaper är att det förekommer individuella skillnader i hur man reagerar på tiopurinläkemedel, beroende på vilken typ av TPMT-protein man har anlag för. Det gör doseringen av tiopuriner komplicerad. En patient som har gener för en lågaktiv TPMT-variant riskerar att överdoseras, vilket kan leda till att immunförsvaret slås ut helt. En patient som har högaktivt TPMT-protein bildar istället stora mängder inaktiverade läkemedelsmolekyler, vilka kan skada levern vid höga halter. För att göra behandling med tiopurinläkemedel säkrare görs rutinmässigt en kontroll av vilken typ av TPMT-protein patienten har, och därefter skräddarsys doseringen för varje patient. Man övervakar också patienten med regelbundna provtagningar för att kontrollera att mängden läkemedel är på rätt nivå. Biokemin som hjälpmedelI vår forskning studerar vi utvalda TPMT-varianter för att förklara vad som orsakar deras olika biofysikaliska egenskaper. Vi har bland annat kunnat visa att den molekylära strukturen hos en variant, TPMT*6 (Y180F), är lite min...

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Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-l-methionine reveals regulation of TPMT1 and 3C allozymes

Cited by 20 publications

References 39 publications

One amino acid makes a difference–Characterization of a new TPMT allele and the influence of SAM on TPMT stability

One amino acid makes a difference–Characterization of a new TPMT allele and the influence of SAM on TPMT stability

Pharmacogenetic variants in TPMT alter cellular responses to cisplatin in inner ear cell lines

Thiopurine S-methyltransferase - characterization of variants and ligand binding

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