Analysis of the immunological role of Phospholipase D4

描述（由申请人提供）：这是一项研究磷脂酶D4（Pld 4）的R21提案，该磷脂酶D4是一种与经典PLD酶具有同源性的功能未知的新基因。最初选择Pld 4进行研究，因为其表达模式似乎仅限于B细胞，并且其mRNA水平在BCR刺激后在未成熟骨髓B细胞中发生变化。我们现在知道它在某些髓系细胞中表达最多，包括浆细胞样树突细胞。我们最近产生了Pld 4的条件性敲除等位基因。我们发现Pld 4基因敲除小鼠具有几种微妙的表型，免疫系统发生了挑衅性的改变，包括巨噬细胞亚群上MHCII表达的大量上调，边缘区B细胞数量的增加和腹膜B-1细胞的减少。抗体反应会发生微妙的变化。我们已经确定的最引人注目的功能表型是Pld 4缺失小鼠对实验性自身免疫性脑脊髓炎（EAE）的诱导完全耐受。令人兴奋的是，抑制这种酶的作用可能有助于治疗多发性硬化症。Pld 4携带一个保守的磷脂酶D催化结构域，具有活性位点组氨酸、赖氨酸和天冬氨酸（HKD基序），但在其他方面与众所周知的家族成员如PLD 1和2显著不同。尚未确定Pld 4的催化活性。我们提出了一个双管齐下的方法来确定是否推定的活性位点是重要的生物功能，并评估在哪些细胞类型的Pld 4的生物活性是需要其多重作用。这项研究的长期目标是了解这种新蛋白质的生物学和生物化学。 公共卫生相关性：我们最近产生了携带突变的小鼠，该突变在特征不明显的Pld 4基因中，并发现这些小鼠对人类多发性硬化症的小鼠模型完全具有抵抗力。这意味着Pld 4蛋白功能的阐明将通过开发阻断其功能的药物来引领多发性硬化症的新治疗方法。为了解决这个问题，我们将确定蛋白质必须在哪些细胞类型中发挥作用以促进疾病，并测试预测阻止Pld 4作为酶催化剂发挥作用的错义突变是否足以预防疾病。

DESCRIPTION (provided by applicant): This is a R21 proposal to investigate Phospholipase D4 (Pld4) a novel gene of unknown function with homology to classical PLD enzymes. Pld4 was chosen for study initially because its expression pattern appeared to be restricted to B cells and its mRNA levels changed in immature bone marrow B cells upon BCR stimulation. We now know that it is expressed most in certain myeloid lineage cells including plasmacytoid dendritic cells. We have recently generated a conditional knockout allele of Pld4. We find that Pld4 null mice have several subtle phenotypes, with provocative alterations in the immune system, including massive up-regulation of MHCII expression on a subset of macrophages, an increase in marginal zone B cell numbers, and a decrease in peritoneal B-1 cells. Antibody responses are subtly altered. The most striking functional phenotype we have identified is that Pld4 null mice are completely resistant to induction of experimental autoimmune encephalomyelitis (EAE). The exciting implication is that inhibition of the action of this enzyme may facilitate treatment of multiple sclerosis. Pld4 carries a conserved phospholipase D catalytic domain with active site histidine, lysine and aspartic acid (HKD motif), but otherwise differs significantly frm well-known family members such as PLD1 and 2. No catalytic activity has been established for Pld4. We propose a two-pronged approach to determine if the putative active site is important for biological function and to assess in which cell types Pld4 bioactivity is required for its multple effects. The long-term goal of this study is to understand the biology and biochemistry of this novel protein. PUBLIC HEALTH RELEVANCE: We recently generated mice carrying a mutation in the poorly characterized Pld4 gene and found that these mice are completely resistant to a mouse model of human multiple sclerosis. The implication is that elucidation of the function(s) of the Pld4 protein will lead the way to new treatments of multiple sclerosis by development of drugs that block its function. To approach this, we will determine in which cell types the protein must function to promote disease and test if missense mutations that are predicted to prevent Pld4 function as an enzyme catalyst are sufficient to prevent disease.