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发育和动态平衡至关重要。虽然大多数形式的细胞凋亡都是通过激活半胱氨酸天冬氨酸酶来实现的，但已有证据表明存在由凋亡诱导因子(AIF)介导的半胱氨酸天冬氨酸氨基转移酶非依赖性途径。然而，将细胞从caspase非依赖性凋亡中拯救出来的机制尚未确定。Preli是一种线粒体蛋白，它具有LEA(晚期胚胎发生丰富)基序的串联重复序列，可以阻断星形孢子素、肿瘤坏死因子-α和紫外线诱导的细胞凋亡。Preli可以维持线粒体的完整性，阻止细胞色素c的释放，保护细胞免受caspase依赖和非依赖的细胞凋亡。我们的数据还表明，Preli可以阻止AIF从线粒体到细胞核的移位，并抑制DMA碎片。Preli作为凋亡抑制因子的相关性被其功能的两个靶向扰动所证实：(I)其功能LEA基序的缺失和(Ii)在人类细胞中的siRNA沉默。因此，本研究揭示了一种进化上保守的机制，即同时反对caspase依赖和独立的细胞凋亡。其具体目的是：1.评估Preli在控制细胞凋亡中的作用。我们将通过酶活性的测量和荧光离子结合探针的使用来测试Preli对呼吸链、活性氧(ROS)产生和钙离子通量的影响。为了研究Preli与其他蛋白质的可能相互作用，我们将进行蛋白质谱筛选、酵母双杂交分析、标签蛋白下拉、激酶分析和Western blotting；ii.在体内评估Preli基因过度表达和失活的影响。我们将建立两个不同的小鼠模型：(A)在F增强子(EF)控制下过度表达Preli的转基因小鼠将被用于研究Preli介导的存活是否导致淋巴细胞增殖和增殖增强，(B)将产生组织特异性Preli缺陷小鼠，以研究其基因失活对B淋巴细胞成熟和分化期间存活的影响。通过用表达CD19驱动的CRE重组酶的小鼠饲养携带FLOXPREI基因的小鼠，将获得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.