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UCP 2和UCP 3与2型糖尿病的病因学、代谢综合征的缓解以及活性氧引起的细胞损伤有关。这些功能与UCP 2和3的解偶联作用一致;然而，文献中的数据在确认这种作用方面是混合的。此外，缺乏核苷酸和脂肪酸调节UCP 2和3功能的信息。这些问题需要一个彻底的生化方法，这将被应用在这个项目中，通过测量质子通量催化的纯化，重组UCP 1，2和3，并通过测量质子通量从细胞中分离的线粒体表达大量的UCP 2和3。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UCP 2和3是否解偶联分离的线粒体并重建天然UCP 2和3活性以验证重组蛋白的性质;（3）确定UCP 1、2和3对核苷酸和酰基CoA酯的抑制亲和力;（4）探讨饱和脂肪酸和不饱和脂肪酸在UCP 1、2和3转运机制中的相对作用。对纯化蛋白的研究将使用实验室建立的方案，用于表达、提取、纯化、重建和测定脂质体中UCP 1、2和3的功能。UCP 1作为这些技术有效性的金标准包括在这些研究中;它是目前唯一可获得天然重建活性的UCP。对分离的线粒体的研究将使用已知高表达UCP的组织，如大鼠肺。三碘甲腺原氨酸将用于刺激大鼠骨骼肌中的UCP 3表达，脂多糖将用于刺激脾脏和肝脏中的UCP 2表达。这些研究将为UCP 2和3的功能和调节机制提供新的理解，也将解决有关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.