Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria.
Institute of Biophysics, Johannes Kepler University, Linz, Austria.
Biochim Biophys Acta Biomembr. 2018 Mar;1860(3):664-672. doi: 10.1016/j.bbamem.2017.12.001. Epub 2017 Dec 5.
Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.
线粒体膜解偶联蛋白 3（UCP3）不仅在骨骼肌和心脏中表达，还与 UCP1 一起在棕色脂肪组织（BAT）中表达，促进质子泄漏以支持非颤抖产热。与 UCP1 不同，UCP3 的转运功能和分子机制研究甚少，尽管人们普遍认为 UCP3 类似于 UCP1，可转运质子，被游离脂肪酸（FFAs）激活，被嘌呤核苷酸（PNs）抑制。由于 BAT 中存在两种相似的解偶联蛋白令人惊讶，我们假设 UCP1 和 UCP3 的调节方式不同，这可能导致它们的功能不同。通过结合原子力显微镜和在平面双层膜中重组蛋白的电生理测量，我们比较了 UCPs 与 PNs 之间的蛋白活性水平和键寿命。我们的数据表明，与 UCP1 相反，UCP3 可被所有 PNs 完全抑制，IC50 随 PN 磷酸化程度的降低而增加。突变蛋白实验表明，PN 结合口袋中保守的精氨酸参与了 UCP1 和 UCP3 的不同程度抑制。脂肪酸与结合在 UCP1 上的所有 PNs 竞争，但仅与结合在 UCP3 上的 ATP 竞争。我们确定磷酸盐是 UCP3 和 UCP1 的一种新型抑制剂，它独立于 PNs 发挥作用。高度同源转运蛋白 UCP1 和 UCP3 之间抑制的分子机制差异表明，UCP3 已适应在 BAT 中发挥不同的作用和可能的另一种转运功能。
