Molecular Pathways of Programmed C ell Death And Viral C

体内死亡程序控制着体内细胞的数量和类型。疾病可由低效的细胞死亡或不适当或过度的死亡引起，例如艾滋病期间的人类免疫缺陷病毒(HIV)或SARS期间的SARS-CoV病毒。在这个项目中，我们采用多方面的方法来研究淋巴细胞和其他细胞类型中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)超家族中的死亡诱导细胞表面受体，如TNFR1和CD95/Fas/APO-1。这两种受体都在刺激细胞的凋亡性和非凋亡性死亡方面发挥重要作用，主要是在免疫过程中。有趣的是，这两种受体都可以起到除死亡以外的作用，比如诱导转录因子。我们正在试图了解这些受体是如何刺激细胞内机制的，这种机制导致细胞死亡，而不是其他细胞结果。我们的大部分工作都集中在激活一种名为caspase-8的蛋白酶上，该酶负责调节死亡程序。我们已经确定了两个源于TNFR1和Fas受体的死亡程序，一个依赖于caspase-8，具有凋亡的形态，另一个不依赖于caspase-8，涉及坏死。有趣的是，只有当caspase-8被抑制时，才能观察到后一种死亡程序。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，在非淋巴样细胞中抑制caspase-8可以导致另一种形式的细胞死亡，表现出特殊的细胞膜结构，称为自噬。尽管最初存在争议，但几个实验室现在已经证明，这种形式的死亡对化疗药物杀死肿瘤细胞特别重要。我们现在已经证明，自噬死亡计划的机制是选择性地降解过氧化氢酶，导致活性氧的显着积累，导致细胞损伤和死亡。 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的病毒感染相关的细胞病变中发挥作用。特别是，在感染艾滋病毒后出现艾滋病的一个关键影响是病毒引起的T淋巴细胞死亡。我们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，它们参与了这一过程。为了严格研究这一过程，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，这两种细胞毒基因产物都会导致G2和M期边界的周期停滞。数学模型表明，细胞丢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，以及这可能是如何发生的。与艾滋病毒不同的是，导致SARS的人类冠状病毒SARS-CoV会导致不涉及细胞周期停滞的坏死性细胞死亡。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 3b，它存在于SARS-CoV中，但不存在于其他病理程度较低的人类冠状病毒中。ORF3b的细胞效应是引起高尔基体的戏剧性重组，这对细胞具有致命的影响。我们现在正试图建立一条连接到ORF3b的分子通路，从而导致这种细胞病变效应。

Internal death programs control the number and types of cells in the body. Diseases can result from inefficient cell death or from inappropriate or excessive death such as is 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 focused much of our efforts on 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 damageand death. 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. 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. 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 3b, 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 ORF3b that causes this cytopathic effect.