Functional characterization of a novel evolutionarily conserved inositol hexakisphosphate kinase in mammalian cells

哺乳动物细胞中新型进化保守肌醇六磷酸激酶的功能表征

• 批准号: 419433583
• 负责人: Dr. Debabrata Laha, Ph.D.
• 金额: --
• 依托单位: Medical Research Council Laboratory for Molecular Cell Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419433583?language=en
• 关键词: Functional; characterization; novel; evolutionarily; conserved

Myo-inositol derived signaling molecules come as two different classes of chemical compounds: membrane-resident phosphoinositides and cytosolic inositol polyphosphates. Inositol polyphosphates represent a multifaceted family of signaling molecules comprising the famous calcium release factor Ins(1,4,5)P3 as well as molecules containing a diphospho- moiety known as inositol pyrophosphates. Inositol pyrophosphates control several crucial cellular and physiological processes including chromatin remodeling, apoptosis, mRNA export, and vesicle trafficking. Recent studies have proposed that many of these functions could be attributed to the ability of inositol pyrophosphates to regulate basic metabolism through phosphate homeostasis. The best characterized inositol pyrophosphates are InsP7 and InsP8. In yeast, amoeba and metazoan, the biosynthesis of InsP7 and InsP8 is mediated by two distinct classes of enzymes, Kcs1/IP6K-type and Vip1/PPIP5K-type proteins, respectively. In plants, InsP7 and InsP8 are also present, however no direct homologs of Kcs1/IP6K-type kinases are encoded by plant genomes. Recently, we have identified a new type of InsP6 kinases in plants that are not sequence-related to Kcs1/IP6K-type kinases and can mediate the biosynthesis of InsP7. Interestingly, this novel type of InsP6 kinase is also encoded by the human genome despite the presence of three Kcs1/IP6K-type kinases. The aim of the proposed research is to functionally characterize this novel InsP6 kinase in mammalian cells by employing a series of comprehensive biochemical, biophysical and cell biological techniques. In short, I will explore the inositol polyphosphate kinase activities of this novel enzyme. I will investigate the molecular structure of the InsP6 kinase reaction products using established biochemical and biophysical techniques. Moreover, I will generate knockout mammalian cells of this kinase using CRISPR and will perform metabolic experiments designed to investigate the inositol polyphosphate homeostasis in the loss-of-function cells. Furthermore, I will combine genetic, cell-biological and metabolic experiments to determine the physiological consequences of these kinase deficient cells under several stresses including phosphate starvation. Taken together, the proposed research will help to understand in depth the molecular mechanisms through which inositol pyrophosphates orchestrate several critical cellular processes and to which degree these processes are associated with phosphate homeostasis.

肌醇衍生的信号分子有两类不同的化合物：膜驻留磷酸肌醇和胞质肌醇多磷酸。肌醇多磷酸代表了一个多方面的信号分子家族，包括著名的钙释放因子Ins（1，4，5）P3以及含有被称为肌醇焦磷酸的二磷酸部分的分子。肌醇焦磷酸控制几个关键的细胞和生理过程，包括染色质重塑、细胞凋亡、mRNA输出和囊泡运输。最近的研究提出，这些功能中的许多可以归因于肌醇焦磷酸通过磷酸盐稳态调节基础代谢的能力。最佳表征的肌醇焦磷酸是InsP 7和InsP 8。在酵母、变形虫和后生动物中，InsP 7和InsP 8的生物合成分别由两类不同的酶介导，Kcs 1/IP 6 K型和Vip 1/PPIP 5 K型蛋白。在植物中，InsP 7和InsP 8也存在，但是没有Kcs 1/IP 6 K型激酶的直接同源物由植物基因组编码。最近，我们在植物中鉴定了一种新的InsP 6激酶，其与Kcs 1/IP 6 K型激酶没有序列相关性，并且可以介导InsP 7的生物合成。有趣的是，这种新型InsP 6激酶也由人类基因组编码，尽管存在三种Kcs 1/IP 6 K型激酶。该研究的目的是通过采用一系列全面的生物化学，生物物理学和细胞生物学技术，在哺乳动物细胞中对这种新型InsP 6激酶进行功能表征。总之，我将探索这种新酶的肌醇多磷酸激酶活性。我将使用已建立的生物化学和生物物理技术研究InsP 6激酶反应产物的分子结构。此外，我将使用CRISPR产生这种激酶的敲除哺乳动物细胞，并将进行旨在研究功能丧失细胞中肌醇多磷酸稳态的代谢实验。此外，我将结合联合收割机遗传，细胞生物学和代谢实验，以确定这些激酶缺陷细胞在几种压力下，包括磷酸盐饥饿的生理后果。综上所述，拟议的研究将有助于深入了解肌醇焦磷酸盐编排几个关键细胞过程的分子机制，以及这些过程与磷酸盐稳态相关的程度。

项目成果

ITPK1 mediates the lipid-independent synthesis of inositol phosphates controlled by metabolism

• DOI: 10.1073/pnas.1911431116
• 发表时间: 2019-12-03
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Desfougeres, Yann;Wilson, Miranda S. C.;Saiardi, Adolfo
• 通讯作者: Saiardi, Adolfo