Gaudry Michael J, Bundgaard Amanda, Kutschke Maria, Ostatek Klaudia, Dela Rosa Margeoux A S, Crichton Paul G, Reznick Jane, Jastroch Martin
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Department of Biology-Zoophysiology, Aarhus University, Aarhus, Denmark.
Acta Physiol (Oxf). 2025 Oct;241(10):e70109. doi: 10.1111/apha.70109.
Uncoupling protein 1 (UCP1) is the crucial protein for non-shivering thermogenesis in placental mammals, but the molecular mechanism of thermogenic proton transport is still unknown. Its histidine pair motif (H145 and H147) has been claimed as a critical element for proton translocation, leading to the paradigmatic "cofactor model" of the UCP1 thermogenic mechanism. The histidine pair motif is mutated (H145Q) in the naked mole-rat (NMR, Heterocephalus glaber) UCP1, suggesting disrupted thermogenic function in line with NMR's poor thermoregulatory abilities. Here, we investigated the functionality NMR versus mouse UCP1 to scrutinized the importance of the histidine pair motif.
Respiratory analyses for UCP1 function were performed in isolated brown adipose tissue mitochondria from NMR and mouse. The histidine pair motif of NMR UCP1 was manipulated through mutations, ectopically overexpressed in HEK293 cells and subjected to plate-based respirometry for functional comparison.
Isolated BAT mitochondria of NMRs display guanosine diphosphate-sensitive respiration, indicative of thermogenically competent UCP1. Overexpressed wildtype NMR UCP1 demonstrates proton leak activity comparable to mouse UCP1. Neither restoration of the histidine pair motif nor full ablation of the motif through a double mutation affects UCP1-dependent respiration.
The UCP1 variant of the NMR, a warm-adapted fossorial species, excludes the histidine pair motif as crucial for UCP1 thermogenic function. Collectively, we show that functional investigation into natural sequence variation of UCP1 not only casts new light on the thermophysiology of NMRs but also represents a powerful tool to delineate structure-function relationships underlying the enigmatic thermogenic proton transport of UCP1.
解偶联蛋白1（UCP1）是胎盘哺乳动物非颤抖性产热的关键蛋白，但其产热质子转运的分子机制仍不清楚。其组氨酸对基序（H145和H147）被认为是质子转运的关键元件，从而形成了UCP1产热机制的典型“辅助因子模型”。裸鼹鼠（NMR，Heterocephalus glaber）的UCP1中组氨酸对基序发生了突变（H145Q），这表明其产热功能受到破坏，这与裸鼹鼠较差的体温调节能力一致。在此，我们研究了NMR与小鼠UCP1的功能，以审视组氨酸对基序的重要性。
对来自NMR和小鼠的分离棕色脂肪组织线粒体进行UCP1功能的呼吸分析。通过突变对NMR UCP1的组氨酸对基序进行操作，在HEK293细胞中异位过表达，并进行基于平板的呼吸测定以进行功能比较。
分离的NMR棕色脂肪组织线粒体显示出对鸟苷二磷酸敏感的呼吸作用，表明UCP1具有产热能力。过表达的野生型NMR UCP1表现出与小鼠UCP1相当的质子泄漏活性。组氨酸对基序的恢复或通过双突变对该基序的完全缺失均不影响UCP1依赖性呼吸。
NMR（一种适应温暖环境的穴居物种）的UCP1变体排除了组氨酸对基序对UCP1产热功能的关键作用。总体而言，我们表明对UCP1自然序列变异的功能研究不仅为NMR的热生理学提供了新的线索，而且是描绘UCP1神秘产热质子转运背后结构-功能关系的有力工具。
