Characterization of functional domains in GIMAP5 protein

GIMAP5 蛋白功能域的表征

• 批准号: RGPIN-2016-04349
• 负责人: Ramanathan, Sheela
• 金额: $ 2.26万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662948
• 关键词: Characterization; functional; domains; GIMAP5; protein

***GIMAP (GTPase of the immune associated nucleotide binding protein) family of proteins was discovered following the identification of the gene responsible for the lymphopenic phenotype (lyp) in BB-DP (BioBreeding diabetes-prone) rats by positional cloning. Mature T lymphocytes undergo spontaneous death in the absence of functional GIMAP5 in these rats. It is presumed that the other members of this highly conserved GIMAP family may be involved in the survival of different hematopoietic cell types. However, the mechanisms by which GIMAP family regulates cell survival or other processes are not known. GIMAP family of proteins are characterized by a N-terminal GTPase domain followed by a coiled-coil domain and in certain members, a C-terminal membrane anchor that can associate with intracellular structures such as mitochondria or lysosomes. Research in my laboratory has been aimed at understanding the function of GIMAP5 in T lymphocytes. As the long-term survival of T lymphocytes in the quiescent state is necessary to maintain their numbers in circulation and in lymphoid organs, we hypothesized that GIMAP5 is required for the integration of these survival signals generated through the T cell antigen receptor (TCR) and the interleukin-7 receptor (IL-7R). Using primary T cells from control and GIMAP5 deficient rats and mice, we showed that: (i) TCR and IL-7 mediated signaling pathways are affected in the absence of functional GIMAP5; (ii) GIMAP5 deficiency impairs Ca2+ entry via plasma membrane channels due to the inability of their mitochondria to sequester Ca2+; (iii) GIMAP5 regulates lysosomal Ca2+. ***Based on our preliminary results, we hypothesize that GIMAP5 influences cellular Ca2+ homeostasis by regulating lysosomal Ca2+. ***Research Plan***1) Identification of GIMAP5 domains and the proteins interacting with GIMAP5: Using different mutant constructs of GIMAP5, we will identify the domains involved in the interaction with microtubules and the associated motor proteins.***2) Regulation of calcium homeostasis by GIMAP5: The endo-lysosomal system stores significant amounts of intracellular Ca2+, in addition to the ER and mitochondria. We will characterize the influence of GIMAP5 and the mutant constructs on the lysosomal Ca2+ compartment.***3) Relationship between lysosomal and mitochondrial Ca2+ stores: We posit that in the absence of GIMAP5, lysosomes may retain more Ca2+, accounting for elevated GPN-induced Ca2+ release. We propose that this elevated free-lysosomal Ca2+ store in cell lacking GIMAP5 may also be directly linked to mitochondria. We will follow mitochondrial Ca2+ uptake using Rhod2 and mitotracker as detailed by us. ***Significance: GIMAP5 plays an essential non-redundant role in keeping resting T cells alive. Hence, the proposed study will help in understanding the role of GIMAP5 in the survival of normal lymphocytes. **

* 在通过定位克隆鉴定负责BB-DP（BioBreeding diabetes-prone）大鼠中淋巴细胞减少表型（lyp）的基因后，发现了蛋白质的GIMAP（免疫相关核苷酸结合蛋白的GTdR）家族。在这些大鼠中，成熟T淋巴细胞在功能性GIMAP 5不存在的情况下经历自发死亡。推测该高度保守的GIMAP家族的其他成员可能参与不同造血细胞类型的存活。然而，GIMAP家族调节细胞存活或其他过程的机制尚不清楚。蛋白质的GIMAP家族的特征在于N-末端GTP结构域，随后是卷曲螺旋结构域，并且在某些成员中，C-末端膜锚可以与细胞内结构如线粒体或溶酶体缔合。 我实验室的研究旨在了解GIMAP 5在T淋巴细胞中的功能。由于T淋巴细胞在静止状态下的长期存活是维持其在循环和淋巴器官中的数量所必需的，因此我们假设GIMAP 5是通过T细胞抗原受体（TCR）和白细胞介素-7受体（IL-7 R）产生的这些存活信号的整合所必需的。使用来自对照和GIMAP 5缺陷大鼠和小鼠的原代T细胞，我们表明：（i）TCR和IL-7介导的信号传导途径在功能性GIMAP 5不存在的情况下受到影响;（ii）GIMAP 5缺陷由于其线粒体不能螯合Ca 2+而损害Ca 2+经由质膜通道的进入;（iii）GIMAP 5调节溶酶体Ca 2+。* 基于我们的初步结果，我们假设GIMAP 5通过调节溶酶体Ca 2+来影响细胞Ca 2+稳态。* 研究计划 *1）GIMAP 5结构域和与GIMAP 5相互作用的蛋白质的鉴定：使用GIMAP 5的不同突变体构建体，我们将鉴定参与与微管和相关马达蛋白相互作用的结构域。2)通过GIMAP 5调节钙稳态：除了ER和线粒体之外，内溶酶体系统还储存大量的细胞内Ca 2+。我们将表征GIMAP 5和突变体构建体对溶酶体Ca 2+区室的影响。3)溶酶体和线粒体Ca 2+储存之间的关系：我们认为，在缺乏GIMAP 5的情况下，溶酶体可能保留更多的Ca 2+，解释了GPN诱导的Ca 2+释放升高。我们提出，这种升高的游离溶酶体钙储存在缺乏GIMAP 5的细胞中也可能直接与线粒体有关。 我们将使用Rhod 2和mitotracker跟踪线粒体Ca 2+摄取，如我们详细描述的。* 重要性：GIMAP 5在保持静息T细胞存活方面发挥着重要的非冗余作用。因此，这项研究将有助于了解GIMAP 5在正常淋巴细胞存活中的作用。 **