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。致病性影响可能是由于细胞死亡效率低下或不适当或过度死亡造成的，例如艾滋病期间的人类免疫缺陷病毒（HIV）或SARS期间的SARS冠状病毒造成的死亡。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和其他细胞类型的凋亡和非凋亡性死亡程序的分子机制。我们研究的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TNFR1和CD95/Fas/APO-1。这两种受体在刺激细胞凋亡和非凋亡性死亡中发挥重要作用，主要是在免疫过程中。对于这些可选择的死亡途径是如何与受体信号相关联的，我们知之甚少。有趣的是，这两种受体在死亡之外都有作用，比如诱导转录因子。我们正试图了解这些受体是如何刺激细胞内机制导致细胞死亡而不是其他细胞结果的。我们花了很多精力来了解一种叫做caspase-8的蛋白酶的激活，它调节着死亡程序。我们已经描述了两种来自TNFR1和Fas受体的死亡程序，一种是caspase-8依赖性的，具有凋亡形态，另一种是caspase-8独立的，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控和分子途径是不同的。此外，我们发现抑制非淋巴样细胞中的caspase-8可导致另一种形式的细胞死亡，表现出特殊的细胞质双膜结构，称为自噬。尽管最初存在争议，但一些实验室现在已经表明，这种死亡形式对于化疗药物导致肿瘤细胞死亡尤为重要。我们现在已经证明，自噬死亡程序的机制是过氧化氢酶的选择性降解，导致活性氧的明显过度积累，导致细胞损伤和死亡。此外，我们关注的是在死亡过程中起关键作用的基因。我们已经发现，果蝇的老处女蛋白的人类同源物hSpin对自噬细胞死亡至关重要。我们研究了该蛋白的生化功能，发现它对溶酶体的生物生成和囊泡运输至关重要。特别是，它在自噬结束时溶酶体重溶中起着至关重要的作用。这些研究将揭示非凋亡性死亡机制。

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. Little is known about how these alternative death pathways are entrained to receptor signaling. 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 double 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 particular, it plays a vital role in lysosomal resomation at the end of autophagy. These studies will shed light on nonapoptotic death mechanisms. In parallel, we are exploring how the regulation of cellular death programs may play a role in cytopathicity associated with 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 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. We have also studied how vif causes cell cycle arrest and found that it is a distinctive mechanism from that induced by vpr. We find that vif can alter the nucleocytoplasmic localization of cyclins and cyclin-dependent kinases which leads to disruption of normal cell cycle progression. We continue to explore how HIV-1 alters to cellular machinery to cause the demise of CD4 T cells. 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 ORF3a 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. Preliminary evidence suggests that it has an inimical effect on the normal interferon response against viral infection and we are currently exploring the molecular basis of this effect.