FUNCTION AND REGULATION OF UNCOUPLING PROTEINS 2 AND 3

新发现的解偶联蛋白UCP 2和UCP 3被认为与体重、糖尿病和非颤抖性产热的调节有关。 UCP 2和UCP 3是线粒体蛋白。 正如其名称所示，它们被认为是能量平衡循环中的终端能量耗散者。然而，对它们的运输机制或管理却一无所知。 因此，本项目的第一个目标是确定它们在细菌中基因表达、蛋白质分离和重组成脂质体后的转运功能。我们将确定蛋白质转运的是什么，特别是它们的转运机制是否与体内解偶联一致。 第二个目标是表征核苷酸和长链酰基CoA酯对UCP 2和UCP 3的调节。 我们将研究抑制和结合的家庭的核苷酸，连同pH值和Mg 2 +-依赖性的抑制/结合。 初步结果表明，UCP 1、UCP 2和UCP 3对核苷酸的敏感性不同。因此，第三个目标是使用定点诱变和功能和结合测定来鉴定UCP中对于调节核苷酸结合和抑制至关重要的氨基酸。 最后，验证天然UCP 2和UCP 3在靶组织中功能性表达至关重要。 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UCP 2和UCP 3。 目标1-3中纯化的重组蛋白质的研究将采用离子特异性荧光探针来定量转运。 对目标4中分离的线粒体的研究将采用抗体来验证蛋白质的存在，并采用呼吸和光散射实验来定量解偶联和转运。该项目的意义在于确定UCP 2和UCP 3的生化功能和调节，这是未知的。

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.