Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.
Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.
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UCP1、UCP2 和嵌合体的结构-功能关系：EPR 分析和 UCP2 的视黄酸激活。
DOI：
10.1046/j.1432-1327.2001.01946.x
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Warden,CH
中科院分区：
文献类型：
--
作者：
Chomiki,N;Voss,JC;Warden,CH
解偶联蛋白（UCPs）由三个重复结构域组成，每个结构域大约有100个氨基酸。我们利用UCP1和UCP2的嵌合体和电子顺磁共振（EPR）来研究这些ucp的结构域特异性。问题包括：核苷酸结合对质子运输的影响是否仅由第三个C末端结构域的氨基酸介导，以及前两个结构域的氨基酸是否参与维甲酸或脂肪酸活化？我们首先证实，我们的重构系统产生的UCP1具有已知的特性，如脂肪酸的激活和嘌呤核苷酸对质子运输的抑制。我们的研究结果证实了重组酵母的观察结果，即维甲酸，而不是已知的激活UCP1的脂肪酸，激活UCP2的质子运输，并且这种激活对核苷酸抑制不敏感。我们构建了嵌合体，其中UCP1或UCP2的最后一个结构域被切换，并测试了脂肪酸或视黄酸的激活和核苷酸的抑制。U1U2由mUCP1（氨基酸1-198）和hUCP2（氨基酸211-309）组成。脂肪酸激活质子转运U1U2和GTP介导的抑制。在另一个嵌合构建体U2U1、hUCP2（氨基酸1-210）和mUCP1（氨基酸199-307）中，维甲酸仍然起到激活剂的作用，但对GTP没有抑制作用。使用EPR（一种非常适合分析膜蛋白（如UCPs）结构的方法），我们证实了UCP2与核苷酸结合。EPR数据显示，暴露于ATP后，UCP1和UCP2的结构发生了很大的变化，这意味着UCP2上存在一个假定的核苷酸结合位点。EPR分析还表明，暴露于嘌呤核苷酸后，UCP1/UCP2嵌合体的构象发生了变化。这些数据表明，在UCP2的C端结构域中存在核苷酸结合位点。该结构域仅在融合到UCP1的N端部分（嵌合体U1U2）时才能够抑制质子的转运。因此，参与核苷酸抑制质子运输的残基位于UCP1的两个第一载体基序中。虽然这些结果与先前报道的C端结构域对核苷酸结合的影响一致，但它们也表明，与N端结构域的相互作用是抑制质子运输所必需的。最后，研究结果表明，UCP2等蛋白可能运输质子，尽管它们不负责基础生热或冷诱导生热。
Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C‐terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1–198) and hUCP2 (amino acids 211–309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1–210) and mUCP1 (amino acids 199–307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide‐binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide‐binding site is present in the C‐terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N‐terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C‐terminal domain on nucleotide binding, they also demonstrate that interactions with the N‐terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold‐induced thermogenesis.