Cloning and functional characterization of an uncoupling protein homolog in hummingbirds.
Cloning and functional characterization of an uncoupling protein homolog in hummingbirds.
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DOI：
10.1152/physiolgenomics.2001.5.3.137
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发表时间：
2001-04
影响因子：
4.6
通讯作者：
Claudia R. Vianna;Thilo Hagen;Chen-Yu Zhang;Eric S. Bachman;Olivier Boss;B. Gereben;A. Moriscot;Bradford B. Lowell;J. Bicudo;Antonio C. Bianco
中科院分区：
文献类型：
--
作者：
Claudia R. Vianna;Thilo Hagen;Chen-Yu Zhang;Eric S. Bachman;Olivier Boss;B. Gereben;A. Moriscot;Bradford B. Lowell;J. Bicudo;Antonio C. Bianco
已从燕尾蜂鸟（Eupetomena macroura）中克隆了一个解偶联蛋白（UCP）同源物的cDNA。利用RT-PCR和已知解偶联蛋白同源物保守结构域的引物，从蜂鸟胸肌（飞行肌）中扩增出解偶联蛋白（HmUCP）cDNA。采用RACE方法克隆了开放阅读框的5'和3'端。HmUCP编码区包含915个核苷酸，推导的蛋白质序列由304个氨基酸组成，分别与人UCP 3、UCP 2和UCP 1的同源性分别为72%、70%和55%。在酵母中对这种新蛋白的解偶联活性进行了表征。在此表达系统中，12 CA 5标记的HmUCP融合蛋白的检测富集线粒体馏分中的Western blot。与大鼠UCP 1相似，HmUCP通过摄取荧光电位敏感性染料3 '，3-二己基氧碳菁碘化物降低线粒体膜电位（在全酵母中测量）。HmUCP mRNA主要在骨骼肌中表达，但通过北方印迹分析评估，在心脏和肝脏中也可检测到高水平。将室内温度降低到12-14摄氏度，会触发蜂鸟典型的休眠/复温循环。无论是在胸肌和心脏，HmUCP mRNA水平的1.5- 3.4倍高，在麻痹。总之，这是第一个报告的UCP同源物在鸟类。这些数据表明，HmUCP有可能作为一个UCP的功能，并可能在复温过程中发挥产热作用。
The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.