Molecular Pathways of Programmed C ell Death And Viral Cytopathicity

内部死亡程序在许多疾病中起着重要作用。 致病作用可由无效的细胞死亡或由不适当或过度的死亡引起，例如由艾滋病期间的人类免疫缺陷病毒（HIV）或SARS期间的SAR-CoV病毒引起的死亡。在这个项目中，我们正在采取多方面的方法来研究淋巴细胞以及其他细胞类型的凋亡和非凋亡死亡程序的分子机制。我们研究的一个主要焦点是肿瘤坏死因子受体（TNFR）超家族中诱导死亡的细胞表面受体，如TNFR 1和CD 95/Fas/APO-1。 这两种受体在刺激细胞凋亡和非凋亡性死亡中起重要作用，主要是在免疫过程中。有趣的是，这两种受体除了死亡之外还具有诸如诱导转录因子的作用。我们正试图了解这些受体如何刺激细胞内机制，导致细胞死亡优先于其他细胞结果。我们已经投入了大量的精力来了解一种名为caspase-8的蛋白酶的激活，这种蛋白酶调节死亡程序。我们的特点是两个死亡程序，源自TNFR 1和Fas受体，一个是caspase-8依赖，并具有凋亡形态和其他caspase-8独立，并涉及坏死。有趣的是，后一种死亡程序仅在caspase-8被抑制时观察到。这两种形式的淋巴细胞死亡的调节和分子途径是不同的。此外，我们还发现，在非淋巴细胞中抑制半胱天冬酶-8可导致另一种形式的细胞死亡，表现出称为自噬的特定细胞质膜结构。 尽管最初存在争议，但几个实验室现在已经表明，这种死亡形式对于化疗剂导致的肿瘤细胞死亡特别重要。我们现在已经表明，自噬性死亡程序的机制是选择性降解过氧化氢酶，导致显着的过度积累的活性氧，导致细胞损伤和死亡。此外，我们还关注了在死亡过程中发挥关键作用的基因。 我们已经发现，人类同源的果蝇spinster蛋白，称为hSpin，是必不可少的自噬细胞死亡。 我们已经研究了这种蛋白的生化功能，发现它对适当的溶酶体生物发生和囊泡运输是重要的。 与此同时，我们正在探索细胞死亡程序的调节如何在与艾滋病和SARS病毒感染相关的细胞病变中发挥作用。 特别是，感染HIV后艾滋病发病的一个关键作用是病毒引起的T淋巴细胞死亡。我们已经发现，这种死亡过程是坏死性的，而不是凋亡性的，现在已经确定了两种病毒基因产物，vif和vpr，参与了这一过程。我们已经发现vpr通过与细胞蛋白结合改变细胞周期并促进细胞死亡，这些蛋白在细胞周期进程中起作用。 为了严格地研究这一过程，我们构建了一个数学模型来分析HIV感染过程中组织培养中的细胞死亡。值得注意的是，这两种细胞毒性基因产物在G2和M期的边界处引起细胞周期停滞。数学模型揭示了细胞损失的主要原因是细胞死亡而不是细胞周期停滞。我们正在使用分子遗传学方法来确定细胞周期停滞是否真的会导致细胞死亡以及这是如何发生的。HIV vpr p蛋白是一种小蛋白质（100个氨基酸），除了三个α螺旋之外，没有明显的结构域或酶基序。 我们已经确定，vpr促进有丝分裂调节因子，如细胞周期蛋白B和Cdk 1，和14-3-3蛋白的theta亚型，抑制细胞周期在G2期之间的明显流产复合物的形成。 复合物似乎是由vpr蛋白第三螺旋上的一个特殊疏水补丁成核的。 与HIV相反，引起SARS的人类冠状病毒SARS-CoV引起的坏死性细胞死亡不涉及细胞周期停滞。 我们发现细胞死亡可以追溯到一个新的开放阅读框架，称为ORF 3a，它存在于SARS-CoV中，但不存在于其他病理性较低的人类冠状病毒中。 ORF 3b的细胞效应是引起高尔基体的显著重组，这对细胞具有致死作用。 我们现在正试图建立一个分子途径夹带到ORF 3a，导致这种细胞病变的影响。 到目前为止，我们的数据暗示TGN 38蛋白，主要发现在高尔基体的反式部分作为ORF 3a蛋白的主要直接目标。 进一步的实验将致力于了解SARS如何通过改变高尔基体触发致命事件的机制。 导致ORF 3a致死的是一个叫做ORF 3b的相邻基因。 这种蛋白质在SARS感染过程中从细胞核运输到细胞核，我们正在研究这如何影响细胞的最终命运。

Internal death programs play significant roles in many diseases. Pathogenic effects can result from inefficient cell death or from inappropriate or excessive death such as that caused by the human immunodeficiency virus (HIV) during AIDS or the SAR-CoV virus during SARS. In this project, we are taking a multifaceted approach to studying molecular mechanisms of both apoptotic and nonapoptotic death programs in lymphocytes as well as other cell types. A major focus of our investigations are death-inducing cell surface receptors in the tumor necrosis factor receptor (TNFR) superfamily such as TNFR1 and CD95/Fas/APO-1. Both receptors play an important role in stimulating both apoptotic and nonapoptotic death of cells principally in immune processes. interestingly, both receptors can have effects beside death such as the induction of transcription factors. We are trying to understand how these receptors stimulate the intracellular machinery that causes cell death in preference to other cellular outcomes. We have devoted many of our efforts to understanding the activation of a protease called caspase-8 which regulates the death program. We have characterized two death programs that emanate from TNFR1 and the Fas receptor, one which is caspase-8 dependent and has an apoptotic morphology and the other which is caspase-8 independent and involves necrosis. Interestingly, the latter death program is only observed when caspase-8 is inhibited. The regulation and molecular pathways of these two forms of lymphocyte death are distinct. In addition, we have discovered that inhibition of caspase-8 in non-lymphoid cells can lead to another form of cell death exhibiting particular cytoplasmic membrane structures called autophagy. although initially controversial, several labs have now shown that this form of death is particularly important for the demise of tumor cells by chemotherapeutic agents. We have now shown that the mechanism of autophagic death program is selective degradation of catalase which leads to a marked overaccumulation of reactive oxygen species leading to cellular damage and death. Furthermore, we have focused on genes that play key roles in this process of death. We have found that the human homologue of the Drosophila spinster protein, called hSpin, is essential for autophagic cell death. We have studied the biochemical function of this protein and found that it is important for proper lysosome biogenesis and vesicle trafficking. In parallel, we are exploring how the regulation of cellular death programs may play a role in cytopathicity associated with the virus infections in AIDS and SARS. In particular, a critical effect in the onset of AIDS following infection with HIV is the death of T lymphocytes caused by the virus. We have found that this death process is necrotic rather than apoptotic and have now identified two viral gene products, vif and vpr, that are involved in this process. We have found that vpr alters the cell cycle and promote death by binding to cellular proteins that have a role in cell cycle progression. In order to study this process rigorously we have constructed a mathematical model to analyze cell death in tissue culture during HIV infection. Remarkably, both of these cytotoxic gene products cause says cycle arrest at the boundary of the G2 and M phases. The mathematical model reveals that the principal cause of cell loss is cell death rather than cell cycle arrest. We are using molecular genetic approaches to determine if cell cycle arrest actually causes cell death and how this might come about. The HIV vpr p protein is a small protein (100 amino acids) with no obvious structural domains or enzymatic motifs other than three alpha helices. We have determined that vpr promotes the formation of an apparently abortive complex between mitotic regulators such as CyclinB and Cdk1, and the theta isoform of the 14-3-3 protein which inhibits the cell cycle in the G2 phase. The complex appears to be nucleated by a particular hydrophobic patch on the third helix of the vpr protein. In contrast to HIV, the human coronavirus that causes SARS, SARS-CoV, causes necrotic cell death that does not involve cell cycle arrest. We have found that cell death can be traced to a novel open reading frame, termed ORF 3a, that is present in SARS-CoV but not other less pathological human coronaviruses. The cellular effect of ORF3b is to cause a dramatic reorganization of the Golgi apparatus which has lethal effects on the cell. We are now trying to established a molecular pathway entrained to ORF3a that causes this cytopathic effect. Thus far, our data implicate the TGN38 protein that is primarily found in the trans portion of the Golgi as the main direct target of the ORF3a protein. Further experiments will be directed at understanding the mechanism of how SARS triggers a lethal event through the alteration of the Golgi apparatus. Contributing to the lethality of ORF3a is an adjacent gene called ORF3b. This protein traffics from the nucleus to the mitochondrion during the course of SARS infection and we are studying how this impacts the ultimate fate of the cell.