Diphosphoinositol phosphate function revealed by chemical and genetic approaches

描述（由申请人提供）：细胞能够通过高度调节的信号通路处理有关其环境和内部状态的大量信息。这种信息传递的关键是小分子第二信使，如cAMP、Ca2+和最近出现的磷酸化肌内酯家族。在肌醇磷酸信使的许多衍生物中，有一个有趣的亚群，它具有高能量的二磷酸基团，即磷酸二磷酸肌醇（或PP-IPs），最近被认为与几种细胞功能有关，包括囊泡运输和细胞凋亡。PP-IPs还与异常生理过程有关，包括胰岛素分泌减少和癌症。由于其化学性质复杂，揭示肌醇磷酸的功能存在重大的技术挑战。本研究旨在克服这些障碍，结合遗传和化学方法，阐明PP-IPs的离散信号功能。更具体地说，该奖项的指导阶段旨在从遗传学角度探究PP-IP功能。参与PP-IP生物合成的酶的全基因组定量遗传相互作用图谱将揭示PP-IP与酿酒酵母中所有其他蛋白质的功能联系（目的1）。目的2将评估PP-IP在胰腺3细胞胰岛素分泌方面的功能。在这里，对PP-IP功能的表征可以帮助指导新型抗糖尿病药物的开发。在本奖项独立期间，目标3、4和5将依靠化学探针的发展来研究PP-IP功能。通过开发一种试剂来亲和纯化二磷酸化蛋白，将评估这种新型翻译后修饰的影响（目的3）。PP-IP类似物可以与其蛋白质结合伙伴共价连接，将用于绘制优选的肌醇磷酸结合位点（目的4）。最后，目的5将探索PP-IPs的金属结合特性，这将用于开发用于体内成像的发光探针。通过在目标1和目标2中生成的细胞模型中使用这些工具，将有可能评估PP-IP信号传导的生理相关性。

DESCRIPTION (provided by applicant): Cell are able to process an enormous amount of information about their environment and their internal status via highly regulated signaling pathways. Critical to this information transfer are small molecule second messengers, such as cAMP, Ca2+, and a recently emerging family of phosphorylated inositides. Among the many derivatives of the inositol phosphate messengers is an intriguing subgroup that possesses high energy diphosphate groups, namely the diphosphoinositol phosphates (or PP-IPs), which have recently been connected to several cellular functions, including vesicular trafficking and apoptosis. The PP-IPs have also been linked to abnormal physiological processes including diminished insulin secretion and cancer. Owing to their chemically complex nature, there exist significant techical challenges to uncovering inositol phosphate function. This proposal seeks to overcome these hurdles, using a combination of genetic and chemical methods, to elucidate the discret signaling functions of the PP-IPs. More specifically, the mentored phase of this award is aimed at interrogating PP-IP function genetically. Genome-wide quantitative genetic interaction mapping of the enzymes involved in PP-IP biosynthesis will reveal the functional connections of PP-IPs with all other proteins in S. cerevisiae (aim 1). Aim 2 will evaluate PP-IP function with respect to insulin secretion from pancreatic (3-cells. Here, the characterization of PP-IP function can help guide the development of novel antidiabetics. During the independent period of this award, aims 3, 4, and 5 will rely on the development of chemical probes to study PP-IP function. Through the development of a reagent for the affinity purification of diphosphorylated proteins, the impact of this novel post-translational modification will be evaluated (aim 3). PP-IP analogues that can be covalently linked to their protein binding partners will serve to map the preferred inositol phosphate binding sites (aim 4). Lastly, aim 5 will explore the metal binding properties of the PP-IPs, which will be exploited for the development of luminescent probes for in vivo imaging. By employing these tools in the cell models generated in aims 1 and 2, it will be possible to assess the physiological relevance of PP-IP signaling. Relevance: The diphosphoinositol phosphates are an important class of secondary messengers in the cell, yet they have received little consideration to date. Their function has clearly been linked to several diseases, including the emerging diabetes epidemic. Elucidation of their signaling function will help to develop novel and improved therapeutics against the development to type 2 diabetes.