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In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*8
Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.ORCID-id: 0000-0002-7642-9263
2017 (engelsk)Inngår i: ACS Omega, E-ISSN 2470-1343, Vol. 2, nr 8, s. 4991-4999Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

sted, utgiver, år, opplag, sider
American Chemical Society (ACS), 2017. Vol. 2, nr 8, s. 4991-4999
Emneord [en]
Polymorphisms, protein stability, S-adenosylmethionine, thiopurine S-methyltransferase, anilinonaphtalene sulfonate
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-80187DOI: 10.1021/acsomega.7b00801ISI: 000409924000100OAI: oai:DiVA.org:liu-80187DiVA, id: diva2:546090
Tilgjengelig fra: 2012-08-22 Laget: 2012-08-22 Sist oppdatert: 2020-12-15bibliografisk kontrollert
Inngår i avhandling
1. Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
Åpne denne publikasjonen i ny fane eller vindu >>Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
2012 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press, 2012. s. 71
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1462
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-80194 (URN)978-91-7519-851-4 (ISBN)
Disputas
2012-09-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (svensk)
Opponent
Veileder
Tilgjengelig fra: 2012-08-22 Laget: 2012-08-22 Sist oppdatert: 2013-10-04bibliografisk kontrollert

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