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BETA
Mårtensson, Lars-GöranORCID iD iconorcid.org/0000-0002-7642-9263
Alternative names
Publications (10 of 24) Show all publications
Wennerstrand, P., Blissing, A. T. & Mårtensson, L.-G. (2017). In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*8. ACS Omega, 2(8), 4991-4999
Open this publication in new window or tab >>In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*8
2017 (English)In: ACS Omega, E-ISSN 2470-1343, Vol. 2, no 8, p. 4991-4999Article in journal (Other academic) 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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keywords
Polymorphisms, protein stability, S-adenosylmethionine, thiopurine S-methyltransferase, anilinonaphtalene sulfonate
National Category
Biochemistry and Molecular Biology Medicinal Chemistry Biocatalysis and Enzyme Technology Biophysics
Identifiers
urn:nbn:se:liu:diva-80187 (URN)10.1021/acsomega.7b00801 (DOI)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2018-01-12Bibliographically approved
Lindqvist Appell, M., Mårtensson, L.-G., Almer, S. & Peterson, C. (2015). Nyttan av farmakogenetik för en mer individualiserad behandling: Exemplet tiopuriner vid inflammatorisk tarmsjukdom och barnleukemi. Läkartidningen, 112, 1229-1233, Article ID DF7L.
Open this publication in new window or tab >>Nyttan av farmakogenetik för en mer individualiserad behandling: Exemplet tiopuriner vid inflammatorisk tarmsjukdom och barnleukemi
2015 (Swedish)In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 112, p. 1229-1233, article id DF7LArticle in journal (Refereed) Published
Abstract [en]

Thiopurines are chemotherapeutic drugs used for treatment of inflammatory bowel diseases and childhood leukemia. Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurines. Individuals lacking TPMT are at increased risk for severe side effects when treated with conventional doses of thiopurines. A research group at the division of drug research at Linköping University is studying thiopurine pharmacogenetics. Since the year 2000, the lab has determined the TPMT status in over 12000 individuals, as an aid to decide thiopurine doses before starting treatment. New knowledge of how genetic factors influence thiopurine treatment effect are anticipated to improve the possibilities for individualization of thiopurine therapy.

Place, publisher, year, edition, pages
Stockholm: Läkartidningen, 2015
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-119996 (URN)26126006 (PubMedID)
Available from: 2015-07-01 Created: 2015-07-01 Last updated: 2017-12-04
Niklasson, M., Andrésen, C., Helander, S., Roth, M., Zimdahl Kahlin, A., Lindqvist Appell, M., . . . Lundström, P. (2015). Robust and convenient analysis of protein thermal and chemical stability. Protein Science, 24(12), 2055-2062
Open this publication in new window or tab >>Robust and convenient analysis of protein thermal and chemical stability
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2015 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, no 12, p. 2055-2062Article in journal (Refereed) Published
Abstract [en]

We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

Place, publisher, year, edition, pages
WILEY-BLACKWELL, 2015
Keywords
protein stability; thermal denaturation; chemical denaturation; circular dichroism; fluorescence; curve fitting; protein stability software; protein denaturation software
National Category
Chemical Sciences Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-124648 (URN)10.1002/pro.2809 (DOI)000368292000014 ()26402034 (PubMedID)
Note

Funding Agencies|Swedish Research Council [2012-5136]; LiU Cancer

Available from: 2016-02-08 Created: 2016-02-08 Last updated: 2017-11-30
Wennerstrand, P., Mårtensson, L.-G., Söderhäll, S., Lindqvist Appell, M. & Zimdahl, A. (2013). Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia. European Journal of Clinical Pharmacology, 69(9), 1641-1649
Open this publication in new window or tab >>Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia
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2013 (English)In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 69, no 9, p. 1641-1649Article in journal (Refereed) Published
Abstract [en]

Purpose

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

Methods

Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

Results

Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

Conclusions

Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2013
Keywords
Leukaemia, 6-mercaptopurine, methotrexate, pharmacogenetics, thiopurine s-methyltransferase
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80190 (URN)10.1007/s00228-013-1521-9 (DOI)000323429900003 ()
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-12-07Bibliographically approved
Lindqvist Appell, M., Wennerstrand, P., Peterson, C., Hertervig, E. & Mårtensson, L.-G. (2010). Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene. Pharmacogenetics and genomics, 20(11), 700-707
Open this publication in new window or tab >>Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene
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2010 (English)In: Pharmacogenetics and genomics, ISSN 1744-6880, Vol. 20, no 11, p. 700-707Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

Keywords
pharmacogenetics; protein stability; right-angle light scattering; single nucleotide polymorphism; thiopurine methyltransferase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-60741 (URN)10.1097/FPC.0b013e3283402ee4 (DOI)20881512 (PubMedID)
Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2015-05-29
Andrésen, C., Jalal, S., Aili, D., Wang, Y., Islam, S., Jarl, A., . . . Sunnerhagen, M. (2010). Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance. Protein Science, 19(4), 680-692
Open this publication in new window or tab >>Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
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2010 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 4, p. 680-692Article in journal (Refereed) Published
Abstract [en]

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

Place, publisher, year, edition, pages
Cold Spring Harbor Laboratory Press, 2010
Keywords
DNA-binding protein, stability, efflux gene regulator, multidrug resistance, MarR family, Biacore, analytical ultracentrifugation, circular dichroism, fluorescence, real-time PCR
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-54849 (URN)10.1002/pro.343 (DOI)000276274900006 ()
Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-12-12Bibliographically approved
Wennerstrand, P., Mårtensson, L.-G., Dametto, P., Hennig, J., Skoglund, K. & Peterson, C. (2010). Different mechanisms behind low enzyme activity in vivo of two different variants of Thiopurine S-methyltransferase, TPMT. Paper presented at 35th Congress of the Federation-of-European-Biochemical-Societies, Gothenburg, Sweden, June 26-July 01, 2010. The FEBS Journal, 277(Suppl. 1), 257-258
Open this publication in new window or tab >>Different mechanisms behind low enzyme activity in vivo of two different variants of Thiopurine S-methyltransferase, TPMT
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2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no Suppl. 1, p. 257-258Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

In treatment of acute lymphoblastic leukemia and inflammatorybowel disease (IBD) thiopurines such as azathioprine and 6-mercaptopurineare used. All of these drugs are prodrugs and are, inthe cell, converted to 6-thioguanines (6-TGNs) and incorporatedinto DNA or inhibiting purine synthesis. A key enzyme for thisregulation is the cytosolic enzyme thiopurine S-methyltransferase(TPMT). This enzyme degrades azathioprine and 6-mercaptopurineto methylmercapto-purine and thereby reduces the bioavailabilityof the 6-TGNs incorporated into DNA. TPMT is apolymorphic enzyme with at least 29 different allelic variantsknown today and is one of the more classical examples of pharmacogeneticswhere the TPMT enzyme activity of the allelic variantsis directly correlated to the clinical dosages of the thiopurines, with a 10–15 fold dosage reduction for an allelic variantwith low TPMT enzyme activity. Even though TPMT is awell studied protein. Many studies have been performed in yeast‘‘suspensions’’ and not on pure protein solutions. It has beenspeculated and in a few cases shown that the reason for the lowactivity for most of the allelic variants is mainly due to the lowstability and/or tendency to aggregate. The mutations in thisstudy TPMT *2 (A80P) and TPMT * 5 (L49S) are both situatedat a distance far from the active site, however the enzyme activitiesare severely affected at 37°C. Preliminary results, using a repertoireof techniques such as CD, fluorescence and limitedproteolysis experiments suggest two different mechanisms for thelow enzyme activity at a temperature corresponding to in vivo conditions.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-58962 (URN)000278565100897 ()
Conference
35th Congress of the Federation-of-European-Biochemical-Societies, Gothenburg, Sweden, June 26-July 01, 2010
Available from: 2010-09-03 Created: 2010-09-03 Last updated: 2017-12-12Bibliographically approved
Lindqvist Appell, M., Wennerstrand, P., Skoglund, K., Lars-Göran, M., Hertervig, E. & Peterson, C. (2009). Explaining TPMT genotype/phenotype discrepancy by identification of a novel sequence variant, TPMT*27. In: 13th International Symposium on Purine and Pyrimidine metabolism in man.
Open this publication in new window or tab >>Explaining TPMT genotype/phenotype discrepancy by identification of a novel sequence variant, TPMT*27
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2009 (English)In: 13th International Symposium on Purine and Pyrimidine metabolism in man, 2009Conference paper, Published paper (Refereed)
Abstract [en]

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21181 (URN)
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2013-10-04
Almstedt, K., Mårtensson, L.-G., Carlsson, U. & Hammarström, P. (2008). Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.. Biochemistry, 47(5), 1288-1298
Open this publication in new window or tab >>Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed) Published
Abstract [en]

Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

Place, publisher, year, edition, pages
Washington: ACS, 2008
Keywords
mutant mapping, carbonic anhydrase, misfolding, loss-of-function, molten globule
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-21056 (URN)10.1021/bi701720p (DOI)
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
Höst, G., Mårtensson, L.-G. & Jonsson, B.-H. (2006). Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 1764(10), 1601-1606
Open this publication in new window or tab >>Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
2006 (English)In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, ISSN 1570-9639, Vol. 1764, no 10, p. 1601-1606Article in journal (Refereed) Published
Abstract [en]

The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

Keywords
Carbonic anhydrase, Hydrolysis, Specificity, Mutagenesis, Protein engineering, Rational design
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-12859 (URN)10.1016/j.bbapap.2006.07.010 (DOI)
Note
Original Publication: Gunnar Höst, Lars-Göran Mårtensson and Bengt-Harald Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, 2006, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, (1764), 10, 1601-1606. http://dx.doi.org/10.1016/j.bbapap.2006.07.010 Copyright: Elsevier http://www.elsevier.com/ Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2013-10-04
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ORCID iD: ORCID iD iconorcid.org/0000-0002-7642-9263

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