Characterization of functional domains in GIMAP5 protein

GIMAP(免疫相关核苷酸结合蛋白GTPase)家族蛋白是在通过定位克隆鉴定BB-DP大鼠淋巴细胞减少表型(LYP)基因后发现的。在这些大鼠中，成熟的T淋巴细胞在缺乏功能的GIMAP5的情况下发生自发死亡。推测这个高度保守的GIMAP家族的其他成员可能参与了不同类型造血细胞的生存。然而，GIMAP家族调控细胞存活或其他过程的机制尚不清楚。GIMAP蛋白家族的特征是由N-末端的GTP酶结构域和卷曲的螺旋结构域组成，在某些成员中，C-末端的膜锚定可以与线粒体或溶酶体等细胞内结构相结合。我实验室的研究旨在了解GIMAP5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对于维持它们在循环和淋巴器官中的数量是必要的，我们假设GIMAP5是通过T细胞抗原受体(TCR)和白介素7受体(IL-7R)产生的这些生存信号的整合所必需的。利用正常对照和GIMAP5缺陷大鼠和小鼠的原代T细胞，我们发现：(I)TCR和IL-7介导的信号通路在缺乏功能的GIMAP5的情况下受到影响；(Ii)GIMAP5缺乏会由于线粒体不能隔离钙而损害质膜通道的钙内流；(Iii)GIMAP5调节溶酶体的钙离子。根据我们的初步结果，我们假设GIMAP5通过调节溶酶体的钙离子而影响细胞内钙的动态平衡。研究计划1)GIMAP5结构域和与GIMAP5相互作用的蛋白质的鉴定：使用不同的GIMAP5突变结构，我们将确定参与与微管和相关运动蛋白相互作用的结构域。2)GIMAP5对钙稳态的调节：除了内质网和线粒体外，内溶酶体系统还存储大量的细胞内钙离子。我们将研究GIMAP5和突变构建体对溶酶体内钙离子区划的影响。3)溶酶体和线粒体钙库之间的关系：我们假设在没有GIMAP5的情况下，溶酶体可能保留了更多的钙离子，这是GPN诱导的钙释放增加的原因。我们认为，在缺乏GIMAP5的细胞中，这种升高的游离溶酶体钙存储也可能与线粒体直接相关。我们将使用Rhod2和MitoTracker跟踪线粒体的钙摄取，正如我们详细介绍的那样。意义：GIMAP5在保持静息T细胞存活方面发挥着基本的非冗余作用。因此，这项拟议的研究将有助于理解GIMAP5在正常淋巴细胞存活中的作用。

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 Plan1) 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.