Studies of germinal center B cell survival

描述（由申请人提供）：细胞存活和凋亡之间的协调平衡对于发育和稳态至关重要。虽然大多数形式的细胞凋亡通过激活半胱天冬酶发生，但已经出现了凋亡诱导因子（AIF）介导的半胱天冬酶非依赖性途径的证据。然而，拯救细胞免于半胱天冬酶非依赖性凋亡的机制尚未确定。本研究的重点是PRELI，一种线粒体蛋白，具有串联重复的莱亚（晚期胚胎发生丰富）基序，它可以消除星形孢菌素，TNF-α和紫外线照射诱导的细胞凋亡。PRELI可维持线粒体的完整性，阻止细胞色素c的释放，保护细胞免于caspase依赖性和非依赖性凋亡。我们的数据还表明，PRELI阻止AIF从线粒体易位到细胞核，并抑制DMA片段化。PRELI作为细胞凋亡抑制因子的相关性通过其功能的两种靶向干扰得到证实：（i）其功能性莱亚基序的缺失和（ii）人细胞中的siRNA沉默。因此，本研究揭示了一种进化上保守的机制，同时反对半胱天冬酶依赖性和独立的细胞凋亡。具体目标是：一。探讨PRELI在细胞凋亡调控中的作用。我们将通过测量酶活性和使用荧光离子结合探针来测试PRELI对呼吸链、活性氧（ROS）产生和Ca 2+通量的影响。为了研究PRELI与其他蛋白质的相互作用，我们将进行蛋白质分析筛选，酵母双杂交分析，标记蛋白质下拉，激酶测定和蛋白质印迹; II.评估PRELI基因过表达和失活的体内效应。我们将产生两种不同的小鼠模型：（a）将产生在f增强子（Ef）控制下过表达PRELI的转基因小鼠，以研究PRELI介导的存活是否导致增强的淋巴细胞扩增和增殖，（B）将产生组织特异性PRELI缺陷小鼠，以研究其基因失活对B淋巴细胞在其成熟和分化期间存活的后果。将通过使携带floxed PRELI基因的小鼠与表达CD 19驱动的Cre重组酶的小鼠交配来获得B细胞靶向缺失。

DESCRIPTION (provided by applicant): Coordinate balance between cell survival and apoptosis is crucial for development and homeostasis. While most forms of apoptosis occur through the activation of caspases, evidence of apoptosis inducing factor (AIF)-mediated caspase-independent pathways has emerged. However, the mechanisms that rescue cells from caspase-independent apoptosis have not been determined. This study focuses on PRELI, a mitochondrial protein that possesses tandem repeats of the LEA (late embryogenesis abundant) motif, which can abrogate apoptosis induced by staurosporine, TNF-a and UV irradiation. PRELI can maintain the integrity of mitochondria, prevent cytochrome c release and protect cells from caspase-dependent and independent apoptosis. Our data also show that PRELI prevents AIF translocation from the mitochondria to the nucleus and suppress DMA fragmentation. The relevance of PRELI as a suppressor of apoptosis was substantiated by two targeted perturbations of its function: (i) deletion of its functional LEA motif and (ii) siRNA silencing in human cells. The present study thus reveals an evolutionarily conserved mechanism that concomitantly opposes caspase-dependent and independent apoptosis. The specific aims are: I. To assess the role of PRELI in the control of apoptosis. We shall test the effect of PRELI on the respiratory chain, reactive oxygen species (ROS) production and Ca2+ fluxes by measurements of enzyme activity and the use of fluorescent ion-binding probes. To investigate putative interactions of PRELI with other proteins, we shall conduct protein-profiling screens, yeast two-hybrid analysis, tagged-protein pull-downs, kinase assays, and western blottings; II. To assess in vivo the effects of PRELI gene overexpression and inactivation. We shall generate two distinct mouse models: (a) Transgenic mice overexpressing PRELI under the control of the f enhancer (Ef) will be produced to investigate whether PRELI-mediated survival leads to enhanced lymphocyte expansion and proliferation, (b) Tissue-specific PRELI-deficient mice will be generated to investigate the consequences of the inactivation of its gene on B lymphocyte survival during their maturation and differentiation. The B cell-targeted deletion will be obtained by breeding mice carrying the floxed PRELI gene with mice expressing CD19-driven Cre-recombinase.