Function and Regulation of Uncoupling Proteins 2 and 3

描述(由申请人提供)：体内证据表明，解偶联蛋白UCP2和UCP3与2型糖尿病的病因、代谢综合征的缓解以及减少由活性氧引起的细胞损伤有关。这些功能与UCP2和UCP3的解偶联作用是一致的；然而，文献中的数据在证实这种作用时混杂在一起。此外，关于核苷酸和脂肪酸对UCP2和UCP3功能调节的信息还很缺乏。这些问题需要一种彻底的生化方法，该方法将应用于本项目，通过测量纯化的、重组的UCP1、2和3催化的质子通量和从高表达UCP2和3的细胞分离的线粒体的质子通量。具体目的是(1)鉴定激活UCP介导的解偶联的生物化合物；(2)确定天然的UCP2和3是否解偶联分离的线粒体并重建天然的UCP2和3的活性以验证重组蛋白的性质；(3)确定UCP1、2和3对核苷酸和酰基A酯的抑制亲和力；(4)探讨饱和脂肪酸和不饱和脂肪酸在UCP1、UCP2和UCP3转运机制中的相对作用。对纯化蛋白的研究将采用实验室建立的UCP1、UCP2和UCP3脂质体的表达、提取、纯化、重组和功能测定的方法。UCP1被纳入这些研究，作为这些技术有效性的黄金标准；它是目前唯一可以获得天然重构活性的UCP。对分离线粒体的研究将使用已知的高表达UCP的组织，如大鼠肺。三碘甲腺原氨酸将用来刺激大鼠骨骼肌UCP3的表达，内毒素将用来刺激脾和肝脏的UCP2表达。这些研究将对UCP2和UCP3的功能和调控机制提供新的理解，并将解决已提出的关于这些蛋白质的机制问题。

DESCRIPTION (provided by applicant): In vivo evidence implicates uncoupling proteins UCP2 and UCP3 in the etiology of type 2 diabetes, in the mitigation of metabolic syndrome, and in reducing cellular damage due to reactive oxygen species. These functions are consistent with an uncoupling role for UCP2 and 3; however data in the literature are mixed in confirming such a role. Moreover, information on regulation of UCP2 and 3 function by nucleotides and fatty acids is lacking. These problems require a thorough biochemical approach, which will be applied in this project by measuring proton flux catalyzed by the purified, recombinant UCP1, 2 and 3 and by measuring proton flux in mitochondria isolated from cells expressing high amounts of UCP2 and 3. The specific aims are (1) To identify biological compounds that activate UCP-mediated uncoupling; (2) To determine whether native UCP2 and 3 uncouple isolated mitochondria and to reconstitute native UCP2 and 3 activity to validate the properties of the recombinant proteins; (3) To determine inhibitory affinities of UCP1, 2 and 3 for nucleotides and acyl CoA esters; (4) To investigate the relative roles of saturated and unsaturated fatty acids in the transport mechanisms of UCP1, 2 and 3. Studies on the purified proteins will use protocols established in the laboratory for expression, extraction, purification, reconstitution, and assay of UCP1, 2 and 3 function in liposomes. UCP1 is included in these studies as a gold standard for the validity of these techniques; it is the only UCP for which native reconstitutive activity is presently available. Studies on isolated mitochondria will use tissues known to express UCP in high amounts, such as rat lung. Triiodothyronine will be used to stimulate UCP3 expression in rat skeletal muscle, and lipopolysaccharide will be used to stimulate UCP2 expression in spleen and liver. These studies will provide new understanding of the mechanisms of UCP2 and 3 function and regulation and will also resolve mechanistic questions that have been raised about these proteins.