Functional analysis of Phospholipase D4

描述（由申请人提供）：这是一份R21申请，旨在研究磷脂酶D4的生物化学和遗传学，磷脂酶D4是一种功能未知的新基因，与经典的磷脂酶D基因同源。相关家族成员蛋白，如PLD1和PLD2将磷脂酰胆碱（PC）转化为磷脂酸，释放胆碱，这一反应对细胞信号传导和膜动力学都有影响。PLD4携带PLD1和2的关键活性位点氨基酸，但缺乏典型的磷脂酶D活性。更多的远亲家庭成员具有核酸酶或dna -酪氨酸磷酸二酯酶活性。在痘病毒中发现了与PLD4特别接近的同源物。PLD4在多种抗原呈递细胞中表达，包括B细胞，在髓细胞的内体区室中表达水平最高，尤其是浆细胞样树突状细胞。在T细胞中也可以看到非常低但可检测的表达水平。我们最近产生了Pld4的条件敲除等位基因，并开始分析Pld4生殖系缺失小鼠。在3个月大时，这些小鼠已经表现出多器官炎症、脾肿大、B细胞发育和胸腺选择可能存在缺陷，以及激活和记忆B细胞和T细胞数量过多的迹象。我们假设PLD4通过调节内体功能在抗原呈递中发挥作用。为了了解Pld4缺乏如何导致免疫表型，我们建议在选定的组织和细胞类型中表征缺乏Pld4的小鼠。第二个目标是使用各种生化方法（包括“脂质组学”筛选）确定PLD4的酶活性。公共卫生相关性：这是一项R21探索性拨款提案，旨在阐明一种名为Pld4的新基因的功能。我们在老鼠身上突变了这个基因，发现突变体在许多器官中产生了严重的炎症，包括肺、唾液腺、肝脏和肾脏，这表明免疫系统失调。然而，Pld4必须发挥作用以避免疾病的关键细胞类型尚不清楚，Pld4基因产物所起的生化作用也不清楚，尽管它与细胞内小泡的调节或形成有关。这个项目将试图阐明这些问题。由于人类携带与小鼠几乎相同的基因，我们的研究与了解人类基因和可能由Pld4缺乏或失调引起的疾病直接相关。

DESCRIPTION (provided by applicant): This is an R21 proposal to investigate the biochemistry and genetics of Phospholipase D4, a novel gene of unknown function with homology to classical phospholipase D genes. Related family member proteins, such as PLD1 and PLD2 convert phosphatidyl choline (PC) to phosphatidic acid, releasing choline, a reaction that has effects on both cell signaling and membrane dynamics. PLD4 carries key active site amino acids of PLD1 and 2, but lacks classical phospholipase D activity. More distantly related family members have nuclease or DNA-tyrosine phosphodiesterase activity. Particularly close homologues to PLD4 are found in pox viruses. PLD4 is expressed in a variety of antigen presenting cells, including B cells, with highest levels in endosomal compartments of myeloid cells, especially plasmacytoid dendritic cells. Very low, but detectable, levels of expression are also seen in T cells. We have recently generated a conditional knockout allele of Pld4 and have begun the analysis of Pld4 germline null mice. At three months of age these mice already exhibit evidence of multiorgan inflammation, splenomegaly, possible defects in B cell development and thymic selection, and excess numbers of activated and memory B and T cells. We hypothesize that PLD4 plays a role in antigen presentation through the regulation of endosomal function. To understand how Pld4 deficiency causes the immunological phenotype we propose to characterize mice lacking Pld4 in selected tissues and cell types. A second goal is to determine the enzymatic activity of PLD4 using a variety of biochemical approaches, including "lipidomics" screens. PUBLIC HEALTH RELEVANCE: This is a R21 exploratory grant proposal to elucidate the function of a novel gene called Pld4. We have mutated this gene in mice and found that the mutants develop severe inflammation in many organs, including lung, salivary gland, liver and kidney, suggesting dysregulation of the immune system. However, the key cell types in which Pld4 must function to avoid disease are not known, nor is it clear what biochemical role the Pld4 gene product plays, though it is implicated in the regulation or formation of small vesicles within cells. This project will attempt to shed light on these issues. Because human beings carry a nearly identical gene to that of mouse, our studies have direct relevance to the understanding of the human gene and possible diseases caused by Pld4 deficiency or dysregulation.