Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis
Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis
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DOI：
10.1016/j.dnarep.2010.03.008
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发表时间：
2010-06-04
期刊：
影响因子：
3.8
通讯作者：
Mazzei, Filomena
中科院分区：
文献类型：
--
作者：
D'Agostino, Vito G.;Minoprio, Anna;Mazzei, Filomena
MUTYH DNA糖基化酶特异性地去除与氧化嘌呤8-氧代-7，8-二氢鸟嘌呤（8-oxoG）相对的通过复制聚合酶错误掺入的腺嘌呤。由于未修复的8-oxoG：A错配，缺陷的蛋白质活性导致G > T颠换的积累。在人类中，MUTYH种系突变与家族性腺瘤性息肉病和结直肠癌易感性的隐性形式（MUTYH相关息肉病，MAP）相关。在这里，我们研究了在MAP患者中鉴定的MUTYH变体R171 W、E466 del、137 insIW、Y165 C和G382 D的修复能力。在从细菌系统中表达和纯化人类蛋白质后，我们通过标准糖基化酶测定研究了MUTYH对8-oxoG：A底物的切割能力。我们首次采用表面等离子体共振（SPR）技术实时记录野生型和MUTYH变体与8-oxoG：A DNA底物的缔合/解离。与野生型蛋白相比，R171 W、E466 del和Y165 C变体对底物的结合亲和力严重降低，而137 insIW和G382 D突变体仅表现出轻微降低，主要是由于缔合速率较慢。这种减少的结合总是与糖基化酶活性的损害有关，腺嘌呤去除在R171 W、E466 del和Y165 C中被完全废除，而在137 insIW和G382 D中仅部分减少。我们的研究结果表明，SPR分析是合适的，以确定有缺陷的酶的行为，即使当突变蛋白质显示轻微的改变，底物识别。(C)2010 Elsevier B. V.保留所有权利。
The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G > T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition. (C) 2010 Elsevier B.V. All rights reserved.