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Thiopurine S-methyltransferase - characterization of variants and ligand binding

機(jī)譯：硫嘌呤S-甲基轉(zhuǎn)移酶-變異體和配體結(jié)合的表征

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摘要

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.
機(jī)譯：硫嘌呤S-甲基轉(zhuǎn)移酶（TPMT）屬于結(jié)構(gòu)相關(guān)蛋白的I類S-腺苷甲硫氨酸依賴性甲基轉(zhuǎn)移酶（SAM-MT）超家族。該大蛋白家族成員的共同點(diǎn)是使用S-腺苷甲硫氨酸（SAM）作為甲基供體的甲基化反應(yīng)的催化作用，盡管SAM-MT可以作用于多種不同的底物并具有許多生物學(xué)上重要的功能。雖然TPMT的天然功能尚不清楚，但該酶與硫嘌呤的代謝有關(guān)，硫嘌呤是一類用于治療免疫相關(guān)疾病的藥物。具體而言，TPMT甲基化可使硫嘌呤及其代謝中間體失活，從而降低了臨床治療的效率并增加了不良副作用的風(fēng)險(xiǎn)。進(jìn)一步使事情復(fù)雜化的是，TPMT是一種多態(tài)性酶，迄今已知有40多種天然存在的變體，其中大多數(shù)對(duì)硫嘌呤的甲基化活性降低。因此，TPMT介導(dǎo)的硫嘌呤失活存在個(gè)體差異，在臨床治療之前必須調(diào)整給藥劑量以避免有害的副作用。盡管TPMT的臨床相關(guān)性已得到很好的確立，但很少有研究調(diào)查變異蛋白甲基化活性降低的分子原因。本文提出了兩種變異蛋白TPMT * 6（Y180F）和TPMT * 8（R215H）的生物物理表征結(jié)果。 TPMT * 8的特性與野生型蛋白沒(méi)有區(qū)別，但發(fā)現(xiàn)TPMT * 6不穩(wěn)定。有趣的是，TPMT * 6氨基酸取代不影響變異蛋白的功能或折疊模式。因此，報(bào)道的針對(duì)TPMT * 6的體內(nèi)功能性下降可能是由于蛋白水解降解增加而引起的，這是由于該蛋白質(zhì)變體的穩(wěn)定性降低而不是功能喪失引起的。本文還提供了用于研究TPMT及其變體的新穎方法論方法。首先，介紹了使用8-苯胺基萘-1-磺酸（ANS）探測(cè)TPMT的三級(jí)結(jié)構(gòu)和活性位點(diǎn)完整性的優(yōu)點(diǎn)。 ANS僅以高親和力（KD?0.2μm）和1：1的比例結(jié)合到TPMT的原始狀態(tài)。 TPMT的穩(wěn)定性通過(guò)ANS的結(jié)合而顯著增加，這表明它與輔助因子SAM的結(jié)構(gòu)相似的腺嘌呤部分共定位。其次，提出了一種基于等溫滴熱法（ITC）的酶活性測(cè)定方法。使用這種方法，已經(jīng)表征了通過(guò)TPMT進(jìn)行6-MP和6-TG甲基化的動(dòng)力學(xué)。

著錄項(xiàng)

  • 作者

    Blissing, Annica;

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  • 年度 2017
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  • 正文語(yǔ)種 eng
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