FUNCTION AND REGULATION OF UNCOUPLING PROTEINS 2 AND 3

新发现的解偶联蛋白UCP2和UCP3被认为参与了体重、糖尿病和非寒颤产热的调节。UCP2和UCP3是线粒体蛋白。正如它们的名字所示，它们被认为是能量平衡循环中的终端能量耗散者。然而，我们对它们的运输机制和调控机制一无所知。因此，本项目的第一个目标是确定它们在细菌中基因表达、蛋白分离和重组为脂质体后的转运功能。我们将确定哪些是由蛋白质运输的，特别是，它们的运输机制是否与体内的解偶联一致。第二个目标是描述核苷酸和长链酰基辅酶a酯对UCP2和UCP3的调控。我们将研究核苷酸家族的抑制和结合，以及抑制/结合的pH和Mg2+依赖性。初步结果表明，UCP1、UCP2和UCP3对核苷酸的敏感性不同。因此，第三个目标是利用位点定向诱变和功能和结合分析，确定ucp中对核苷酸结合和抑制调控至关重要的氨基酸。最后，验证天然UCP2和UCP3在靶组织中是否有功能表达是至关重要的。因此，第四个目标是鉴定和测定来自棕色脂肪、心脏、肾脏、骨骼肌、白色脂肪和淋巴细胞的线粒体中的天然UCP2和UCP3。目标1-3中纯化重组蛋白的研究将使用离子特异性荧光探针来定量转运。目标4中对分离线粒体的研究将使用抗体来验证蛋白质的存在，并使用呼吸和光散射实验来定量解偶联和运输。本项目的意义在于确定未知的UCP2和UCP3的生化功能和调控。

The newly discovered uncoupling proteins, UCP2 and UCP3, are thought to be involved in regulation of body weight, diabetes, and non-shivering thermogenesis. UCP2 and UCP3 are mitochondrial proteins. As indicated by their name, they are thought to be terminal energy dissipators in the energy balance cycle. However, nothing is known about their transport mechanisms or regulation. Accordingly, the first goal of this project is to determine their transport functions following gene expression in bacteria, protein isolation, and reconstitution into liposomes. We will determine what is transported by the proteins, and in particular, whether their transport mechanisms are consistent with uncoupling in vivo. The second goal is to characterize regulation of UCP2 and UCP3 by nucleotides and long-chain acyl CoA esters. We will investigate inhibition and binding of the family of nucleotides, together with the pH- and Mg2+-dependence of inhibition/binding. Preliminary results indicate that UCP1, UCP2 and UCP3 differ in their sensitivity to nucleotides. Accordingly, the third goal is to identify amino acids in the UCPs that are critical for regulation of nucleotide binding and inhibition, using site-directed mutagenesis and assays of function and binding. Finally, it is crucial to verify that native UCP2 and UCP3 are functionally expressed in the target tissues. Thus, the fourth goal is to identify and assay native UCP2 and UCP3 in mitochondria from brown fat, heart, kidney, skeletal muscle, white fat, and lymphocytes. The study of purified, reconstituted proteins in Goals 1-3 will employ ion-specific fluorescent probes to quantitate transport. The study of isolated mitochondria in Goal 4 will employ antibodies to verify the presence of proteins, and respiration and light scattering experiments to quantitate uncoupling and transport. The significance of this project lies in determining the biochemical function and regulation of UCP2 and UCP3, which are unknown.