Elucidating cellular signaling functions of diphosphoinositol polyphosphates

细胞能够通过高度调控的信号通路处理关于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、钙离子和最近出现的磷酸化肌苷家族。在肌醇磷酸信使的众多衍生物中，有一个耐人寻味的亚基具有高能量 二磷酸基团，即二磷酸肌醇磷酸盐(PP。-IPs)，最近被连接到几个细胞功能，包括囊泡运输和细胞凋亡。PP-IP还与异常的生理过程有关，包括胰岛素分泌减少和癌症。 由于其复杂的化学性质，使用标准的细胞生物学技术来揭示肌醇磷酸的功能存在重大的技术挑战。这项建议试图用化学方法克服这些障碍，阐明PP-IP的离散信号功能。在本奖项的独立期内，AIMS的1、2和3将依靠化学探针的发展来研究PP-IP 功能。通过开发一种用于二磷酸化蛋白质亲和纯化的试剂，将评估这种新的翻译后修饰的影响(目标1)。可以共价连接到其蛋白质结合伙伴的PP-IP类似物将用于绘制首选的肌醇磷酸结合位点图(目标2)。最后，目标3将探索PP-IP的金属结合特性，然后将 用于开发用于活体成像的发光探针。 重要的是，来自AIMS 1-3的工具将在指导阶段生成的单元模型中进行评估。遗传相互作用数据将作为一个平台来生成和验证关于PP-IP信号功能的假设。胰岛素瘤细胞是了解PP-IP在胰岛素分泌中作用的关键模型系统。通过将化学试剂与这些基因分析相结合，将有可能评估PP-IP信号的生理相关性。

Cells are able to process an enormous amount of infonnafion 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 (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 technical challenges to uncover inositol phosphate function using standard cell biology techniques. This proposal seeks to overcome these hurdles using chemical methods, to elucidate the discrete signaling functions of the PP-IPs. During the independent period of this award, aims '1,2, and 3 will rely on the deveiopment 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 1). PP-IP analogues that can be covalently linked to their protein binding partners will serve to map the preferred inositol phosphate binding sites (aim 2). Lastly, aim 3 will explore the metal binding properties of the PP-IPs, which vwll then be exploited for the development of luminescent probes for in vivo imaging. Importantly, the tools from aims 1-3 will be evaluated in the cell models that have been generated during the mentored phase. The genetic interaction data will serve as a platform to generate and validate hypotheses about PP-IP signaling functions. The insulinoma cells are a critical model system to understand the role of PP-IPs in insulin secretion. By combining the chemical reagents with these genefic analyses, it will be possible to assess the physiological relevance of PP-IP signaling.