Klingenberg Martin
Institut für physiologische Chemie, Universität München, Schillerstr. 44, 80336 München, Germany.
Biochimie. 2017 Mar;134:19-27. doi: 10.1016/j.biochi.2016.10.012. Epub 2016 Oct 26.
This review focuses on the biochemical work of UCP1 starting from the early observation by Ricquier and Kader in 1976. We entered this field in 1980 with the isolation of native UCP1 and then reported the amino acid sequence structure discovering a strong homology to the ADP/ATP carrier. With the isolated native UCP1 we studied structural and functional features, in particular the complex characteristics of nucleotide binding. A strong pH dependence of binding and herein the differences between diphopho- and triphopho-nucleotides were observed, resulting in the identification of residues which control binding site access by their H dissociation. Newly synthesized fluorescent nucleotide derivatives provided tools to determine a two state nucleotide binding in line with loose and tight UCP1 conformations and H transport inhibition. The slow transition between these states were a notable feature. The reconstitution of isolated UCP1 in vesicles demonstrated that UCP1 protein is in fact the uncoupling factor and not only a nucleotide controlled regulator. The H transport was shown to be electrophoretic with a linear relation to the membrane potential. The dependence of H transport on fatty acids (FA) was characterized and is elaborated here with a view of the experimental conditions of other research groups which had different views of the role of FA in H transport. Furthermore, to explain the contrast of the FA - nucleotide competition between mitochondria and reconstituted system, indirect paths for FA to relieve the inhibition in mitochondria are here proposed, such as a FA induced upward pH shift and a FA induced increase of cardiolipin level around UCP1 since cardiolipin has been found by us to relieve nucleotide binding on isolated UCP1. Recently reported patch clamp results on mitoplasts led to a reformulation of the H transport mechanism of FA in UCP1 in which bound FA shuttles with the carboxyl group between the two membrane sides along the translocation channel outward as FA and inward as FAH. We propose here a modified version, where FA forms an immobile prosthetic group surrounded by the inner and outer gate of the H translocation channel. By alternating opening of the gates FA takes up H from the cytosol side and releases H to the matrix.
本综述聚焦于解偶联蛋白1（UCP1）的生化研究工作，始于1976年里基耶尔（Ricquier）和卡德（Kader）的早期观察。我们于1980年进入该领域，分离出天然UCP1，随后报道了其氨基酸序列结构，发现它与ADP/ATP载体有很强的同源性。利用分离出的天然UCP1，我们研究了其结构和功能特征，特别是核苷酸结合的复杂特性。观察到结合对pH有很强的依赖性，以及二磷酸和三磷酸核苷酸之间的差异，从而确定了通过其H解离来控制结合位点通道的残基。新合成的荧光核苷酸衍生物为确定与UCP1的松散和紧密构象以及H转运抑制相一致的双态核苷酸结合提供了工具。这些状态之间的缓慢转变是一个显著特征。将分离的UCP1重建到囊泡中表明，UCP1蛋白实际上就是解偶联因子，而不仅仅是一个受核苷酸控制的调节因子。H转运被证明是电泳性的，与膜电位呈线性关系。对H转运对脂肪酸（FA）的依赖性进行了表征，并结合其他研究小组对FA在H转运中作用有不同观点的实验条件在此进行阐述。此外，为了解释线粒体和重建系统中FA - 核苷酸竞争的差异，本文提出了FA在线粒体中缓解抑制作用的间接途径，例如FA诱导的pH向上偏移以及FA诱导的UCP1周围心磷脂水平升高，因为我们发现心磷脂可缓解分离的UCP1上的核苷酸结合。最近报道的关于线粒体膜片钳的结果导致了对UCP1中FA的H转运机制的重新表述，即结合的FA通过羧基沿着转运通道在两个膜面之间穿梭，向外为FA，向内为FAH。我们在此提出一个修改版本，其中FA形成一个固定的辅基，被H转运通道的内门和外门包围。通过交替打开门，FA从胞质溶胶侧摄取H并将H释放到基质中。
