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Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 30, 5912-5920 p.Article in journal (Refereed) Published
Abstract [en]

The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012. Vol. 51, no 30, 5912-5920 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80185DOI: 10.1021/bi300377dISI: 000308262600005OAI: oai:DiVA.org:liu-80185DiVA: diva2:546089
Note

funding agencies|Swedish Childhood Cancer Foundation||Swedish Research Council||EMBO||

Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
Open this publication in new window or tab >>Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the treatment of leukemia and inflammatory bowel disease, thiopurines are commonly used drugs. Thiopurine S-methyltransferase (TPMT) is one of the drug metabolizing enzymes responsible of counteracting the formation of TGNs that will be incorporated into the DNA and RNA synthesis and thus induce apoptosis. TPMT is a polymorphic enzyme and to date about 30 different sequence variants have been identified. Individuals who are to be treated with thiopurines are genotyped and/ or phenotyped at the time of diagnosis in order to individualize the treatment, with thiopurine dosage adjusted to the TPMT activity. In the treatment of acute lymphoblastic leukemia (ALL) high-dose methotrexate (MTX) is administered intravenously during the consolidation phase of the therapy and used in lower doses in the other phases of the ALL therapy. In blood samples from 53 children with ALL, we found decreased TPMT enzyme activity after 66 hours infusion of high-dose MTX. TPMT was recombinantly expressed, and the potential binding of MTX to TPMT was investigated by a fluorescence method. This showed that MTX bound to TPMT at relevant plasma concentrations observed in patient samples. At the time of leukemia diagnosis, TPMT activity was not correlated with the genotype for TPMT wild-types, which demonstrates the importance of using genotyping as a golden standard for determination of TPMT status in individuals with haematological malignancies. The low enzyme activity of TPMT*2 and TPMT*5 protein was evaluated by expressing these sequence variants in Escherichia coli (E.coli), and then characterizing them biophysically. Our results showed that TPMT*2 and TPMT*5 in the native state did not bind the extrinsic probe anilinonaphthalene sulfonate (ANS), which shows that the three-dimensional structure is already affected and restructured in that state. Based on these findings, we concluded that ANS can be used to probe the status of the active site. In another study we investigated the characteristics of TPMT*6 and TPMT*8 and found that the cofactor, S-adenosylmethionine (SAM) had a stabilizing effect on those sequence variants and on TPMT wild-type. Analysis of the structure of the TPMT protein by nuclear magnetic resonance (NMR) spectroscopy, enabled partial assignment of the backbone residue, of 64% of the TPMT sequence. Forty residues in TPMT exhibited millisecond dynamics but only 15 of those residues could be assigned, which emphasizes the difficulties involved in determining the three-dimensional structure of TPMT by NMR spectroscopy. In conclusion the present studies contribute to the understanding of the molecular characteristics of TPMT.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 71 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1462
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80194 (URN)978-91-7519-851-4 (ISBN)
Public defence
2012-09-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (Swedish)
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Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2013-10-04Bibliographically approved

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Wennerstrand, PatriciaHennig, JanoschKlingstedt, TheréseSkoglund, KarinLindqvist Appell, Malin

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