Elucidation of the interaction network of diadenosine triphosphate

除了典型的单磷酸、二磷酸和三磷酸外，细胞还含有几种次要的核苷酸。其中，发现了各种二核苷多磷酸（NpnN，n = 2-7）。二核苷多磷酸由两个核苷部分组成，它们在各自的5 ′-羟基上通过多磷酸链连接。这些分子可以在原核细胞以及真核细胞中发现。尽管人们已经知道了50年，但二核苷多磷酸的功能仍然不清楚。研究最多的一种是二腺苷三磷酸（Ap 3A）。其细胞浓度取决于细胞类型和环境因素，在纳摩尔至较低微摩尔范围内变化。强制降解条件下多次报告Ap 3A浓度增加。因此，据推测，Ap 3A作为应激源的细胞信号（警报），因此参与细胞对应激条件的适应过程。然而，它们的确切作用尚未明确。人类中已知的Ap 3A水解酶和唯一经验证的相互作用配偶体是脆性组氨酸三联体蛋白（Fhit）。它由FHIT基因编码，是人类基因组中最脆弱的位点之一。因此，在人癌细胞中通常观察到Fhit表达的丧失及其酶活性的改变。Fhit-Ap 3A复合物被认为是肿瘤抑制活性的活性分子，而不知道进一步的相互作用伙伴。由于有关Ap 3A相互作用组的知识是稀疏的，我们的目标是在这个项目中开发和应用新的手段发现Ap 3A相互作用的合作伙伴。我们将开发和合成不可水解的Ap 3A（nh Ap 3A）类似物，其具有光亲和交联和亲和标签的光敏功能。在与细胞裂解物一起孵育并照射后，光反应性nh Ap 3A类似物将共价结合相互作用的蛋白质，所述相互作用的蛋白质又将用亲和标签标记。标记蛋白富集后，我们将进行亲和纯化，并通过质谱（MS）鉴定蛋白。因此，我们将与康斯坦茨大学的蛋白质组学设施密切合作。因此，我们将确认所鉴定的蛋白质与Ap 3A相互作用的能力。此外，我们的目标是深入了解Fhit的相互作用组，在人类中的Ap 3A的水解酶，和Fhit与Ap 3A的复合物。因此，我们将设计将nh Ap 3A类似物共价连接到Fhit活性位点的方法。Fhit-Ap 3A复合物将用于形成亲和基质，以通过亲和纯化鉴定选择性结合伴侣。由于这些实验将使用Fhit-和Fhit-Ap 3A复合物修饰的基质平行进行，因此将获得对Fhit和Fhit-Ap 3A相互作用组的了解。同样，蛋白质将被鉴定为我的MS，并将研究它们与Fhit和Fhit-Ap 3A的相互作用。

Cells contain several minor nucleotides in addition to the canonical mono-, di- and triphosphates. Among these, various dinucleoside polyphosphates (NpnN, n = 2-7) are found. The dinucleoside polyphosphates consist of two nucleoside moieties, which are linked by a polyphosphate chain at the respective 5'-hydroxyl groups. These molecules can be found in prokaryotic as well as eukaryotic cells. Despite being known for 50 years the functions of dinucleoside polyphosphates are still unclear. One of the most studied one is diadenosine triphosphate (Ap3A). Its cellular concentrations vary dependent on cell type and environmental factors in a nanomolar to a lower micromolar range. An increase of Ap3A concentration was reported several times upon stress conditions. Therefore it was postulated, that Ap3A serve as a cellular signal (alarmones) of stressors and thus be involved in the adaptive processes of cells to stress conditions. However, their exact role is yet to be specified. The known Ap3A hydrolase in humans, and sole verified interaction partner, is the fragile histidine triad protein (Fhit). It is encoded by the FHIT gene, one of the most fragile sites of the human genome. Hence, the loss of Fhit expression and the altering of its enzymatic activity are commonly observed in human cancer cells. The Fhit-Ap3A complex is suggested to be the active molecule for tumor suppressor activity without knowing further interaction partners. Since the knowledge about the Ap3A interactome is sparse we aim at developing and applying new means for the discovery of Ap3A interaction partners within this project. We will develop and synthesize non-hydrolysable Ap3A (nh Ap3A) analogues that bear a light sensitive functionality for photoaffinity crosslinking and an affinity tag. Upon incubation with cell lysates and irradiation, the photoreactive nh Ap3A analogue will covalently bind to interacting proteins, which in turn will be labelled with the affinity tag. After enrichment of the labelled proteins we will conduct affinity purification and the proteins will be identified by mass spectrometry (MS). Hereby, we will closely cooperate with the Proteomics Facility of the University of Konstanz. Thereupon, we will confirm the identified proteins ability to interact with Ap3A. Furthermore, we aim at gaining insights into the interactome of Fhit, the hydrolase of Ap3A in humans, and Fhit in complex with Ap3A. Therefore, we will device means to covalently connect an nh Ap3A analogue to the active site of Fhit. The Fhit-Ap3A complex will be employed for the formation of affinity matrices for the identification of selective binding partners by affinity purification. Since these experiments will be conducted in parallel using Fhit- and Fhit-Ap3A complex-modified matrices insights into the Fhit and Fhit-Ap3A interactome will be obtained. Again, proteins will be identified my MS and their interaction with Fhit and Fhit-Ap3A will be studied.