Role of Polyamines and Hypusine in Nutrient-Induced Beta-Cell Growth and Replication

该项目的灵感来自于这样一个前提，即β细胞外部的信号和底物触发细胞周期进入，并且β细胞中细胞周期的重新激活将为糖尿病患者恢复β细胞质量和功能提供可能性。我们公布的初步数据表明，翻译因子eIF5A作为一种急性反应因子，催化特定mRNAs的翻译，使细胞复制成为可能。引人注目的是，eIF5A是唯一一种含有独特的多胺衍生氨基酸亚硫氨酸的蛋白质，这是其功能所必需的。Hypusine的形成是在翻译后发生的，由鸟氨酸脱羧酶(ODC)和脱氧亚硫氨酸合成酶(DHPS)两种限速酶控制。ODC从精氨酸(以及间接从谷氨酰胺、脯氨酸、蛋氨酸、苯丙氨酸、亮氨酸)产生胞内多胺，DHPS利用多胺亚精胺在eIF5A上形成亚精氨酸。总而言之，我们将ODC/DHPS/eIF5A蛋白称为属于“多胺/下苏氨酸途径”。由于多胺和亚精氨酸的产生可以通过饮食和/或小分子来操纵，它们代表了干预β细胞生长和复制的真实靶点。我们假设，产生多胺和亚精氨酸的途径将营养信号(氨基酸、葡萄糖)连接到信使核糖核酸的翻译，从而实现适应性的β细胞复制。这个多PI R01应用程序的优势是R.Mirmira博士(β细胞中多胺/亚硫氨酸途径的专家)和L.Alonso博士(β细胞复制专家)之间的合作努力；我们的团队在相关专业知识、新型条件基因敲除小鼠和信使核糖核酸翻译评估工具中独一无二地定位于测试这一假设。我们提出了以下3个目标：目的1：阐明多胺/下丘脑通路在肥胖和高血糖模型中调控适应性β细胞增殖的机制。目的2：研究多胺生物合成在β-细胞中对UPR诱导的增殖信号的营养激活把关作用。目的3：揭示eIF5A作为氨基酸和多胺供应生物传感器的特殊功能。我们将使用最先进的成像和细胞生物学技术和新试剂的综合工具箱，包括唯一的odc/dhps/eif5A基因敲除小鼠的集合，揭示在β细胞增殖中的作用，否则是一个谜。因此，这一建议的主要影响是在啮齿动物和人类β细胞复制中多胺/亚精氨酸途径的识别和机制。

This project is inspired by the premise that signals and substrates extrinsic to the β-cell trigger cell cycle entry, and that reactivation of the cell cycle in β-cells would provide the potential for restoring β-cell mass and function in diabetes. Our published and preliminary data suggest that the translation factor eIF5A functions as an acute response factor that catalyzes translation of specific mRNAs, enabling cellular replication. Strikingly, eIF5A is the only protein containing the unique, polyamine-derived amino acid hypusine, which is required for its function. Hypusine formation occurs posttranslationally and is governed by two rate-limiting enzymes, ornithine decarboxylase (ODC) and deoxyhypusine synthase (DHPS). ODC generates intracellular polyamines from arginine (and indirectly from glutamine, proline, methionine, phenylalanine, leucine), and DHPS utilizes the polyamine spermidine to form hypusine on eIF5A. Collectively, we refer to the ODC/DHPS/eIF5A proteins as belonging to the “polyamine/hypusine pathway.” Because polyamine and hypusine productions can be manipulated by diet and/or small molecules, they represent real-world targets to intervene in β-cell growth and replication. We hypothesize that the pathway that generates polyamines and hypusine links nutritional signals (amino acids, glucose) to mRNA translation to enable adaptive β-cell replication. The strength of this Multi-PI R01 application is the collaborative effort between Drs. R. Mirmira (an expert in the polyamine/hypusine pathway in β-cells) and L. Alonso (an expert in β-cell replication); our Team is uniquely positioned with the relevant expertise, novel conditional knockout mice, and mRNA translation assessment tools to test this hypothesis. We propose the following 3 aims: Aim 1: Elucidate the mechanisms by which the polyamine/hypusine pathway governs adaptive β-cell proliferation in models of obesity and hyperglycemia. Aim 2: Characterize how polyamine biosynthesis functions as a nutrient-activated gatekeeper for the proliferative signal induced by the UPR in β-cells. Aim 3: Reveal an unusual function of eIF5A as a biosensor for amino acid and polyamine supply. We will use a comprehensive toolbox of state-of-the-art imaging and cell biology techniques and novel reagents, including the only collection of ODC/DHPS/eIF5A knockout mice, to reveal a role in β-cell proliferation for an otherwise enigmatic pathway. Therefore, the primary impact of this proposal is the identification and mechanisms of the polyamine/hypusine pathway in rodent and human β-cell replication.