Molecular Pathways of Programmed C ell Death And Viral Cytopathicity

内部死亡计划在许多疾病中扮演着重要的角色。致病效应可由低效细胞死亡或不适当或过度死亡引起，如艾滋病期间的人类免疫缺陷病毒(HIV)或SARS期间的SAR-CoV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中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。关于这些替代死亡途径如何与受体信号联系在一起，我们知之甚少。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。我们已经发现，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，表现出特殊的细胞质双膜结构，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，从而导致细胞损伤和死亡。此外，我们还关注了在这一死亡过程中发挥关键作用的基因。 自噬是从人类到酵母的一个进化保守的过程，细胞质蛋白和细胞器被分解，但在自噬结束时，当细胞在自噬细胞死亡和存活之间进行选择时，人们对结果知之甚少。在饥饿期间，控制细胞代谢的营养反应蛋白TOR(雷帕霉素的靶标)被关闭，自噬被激活。双膜自噬小体隔离细胞内的成分，然后与溶酶体融合形成自溶酶体，自溶体分解其内容物以再生营养物质。我们目前对自噬的理解是，它随着自溶体内的货物降解而终止，但自噬是如何由营养物质控制的，以及自溶酶体随后的命运尚不清楚。我们发现，哺乳动物细胞中的mTOR信号在自噬开始时被抑制，但在长期饥饿时重新激活。MTOR的重新激活依赖于自溶酶产物的降解和营养物质的释放。MTOR活性反过来终止自噬并刺激令人印象深刻的原溶酶体小管和囊泡，这些小管和囊泡从自溶酶体中挤出，最终成熟为功能溶酶体。这一过程，我们称之为自噬溶酶体修复(ALR)，恢复了细胞中完整的溶酶体。自噬中的这一进化保守的循环控制着饥饿期间的营养感知和溶酶体内平衡。 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。我们之前发现，这种死亡过程是坏死性的，而不是凋亡的。2012年，我们对人类免疫缺陷病毒1型(HIV-1)的两个主要细胞病变因子进行了研究，即辅助蛋白病毒感染性因子(Vif)和病毒蛋白R(VPR)，它们在细胞周期的G2期抑制细胞周期进展，导致细胞坏死性死亡。虽然Vpr诱导的阻断和相关的T细胞死亡已经被很好地研究了，但Vif阻止G2的分子机制仍然不清楚。为了阐明Vif如何通过诱导G2期停滞来杀死细胞，我们感染了同步化的Jurkat人T细胞，并检测了Vif对G2向有丝分裂相转变的主要细胞周期因子CDK1和CylinB1激活的影响。我们发现，在表达Vif的感染细胞中，CDK1上的抑制性磷酸没有发生特征性的去磷酸化。此外，CDK1和CyclinB1的核转位也受到损害。Vif诱导的细胞周期停滞和细胞毒作用与Vif的表达呈正相关。我们的结论是，Vif通过干扰CDK1-CyclinB1的激活而损害有丝分裂进程，并导致致命的细胞周期中断。 2012年，我们还发现，在自噬过程中，多个溶酶体与自噬小体融合形成自溶酶体，其中细胞质成分被溶酶体水解酶隔离和降解，溶酶体水解酶将产物通过溶酶体外排进入胞浆。在饥饿诱导的自噬之后，溶酶体的动态平衡通过自噬溶酶体重组(ALR)恢复，需要激活“雷帕霉素靶标”(TOR)激酶。Spist(Spin)编码一种类似于糖转运蛋白的溶酶体外排通透酶。自旋突变体在果蝇和人类细胞中积累溶酶体碳水化合物并产生扩大的溶酶体。我们还证明，在长时间饥饿期间，Spin对于mTOR的重新激活和溶酶体的重建是至关重要的。最后，我们证明了SPIN的糖转运蛋白活性是ALR所必需的。这些结果提供了一个详细的分子洞察力，了解在饥饿和其他自噬诱导条件下溶酶体的生物发生是如何进行的。我们还推测，即使在营养充足的条件下，这种机制也可能是正常溶酶体生物发生的基础。

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 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. Autophagy is an evolutionarily conserved process from humans to yeast by which cytoplasmic proteins and organelles are catabolized but very little was known about results at the end of autophagy when cells were selecting between autophagic cell death and survival. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase that controls cellular metabolism, is shut off, and autophagy is activated. Double-membrane autophagosomes sequester intracellular components and then fuse with lysosomes to form autolysosomes, which to catabolize their contents to regenerate nutrients. Ourpresent understanding of autophagy is that it terminates with cargo degradation within autolysosomes, but how autophagy is controlled by nutrients and the subsequent fate of the autolysosome were unknown. We discovered that mTOR signalling in mammalian cells is inhibited during initiation of autophagy, but reactivated during extended starvation. Reactivation of mTOR depends on the degradation of autolysosomal products and release of nutrients. mTOR activity in turn terminates autophagy and stimulates impressive proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes. This process, that we term autophagic lysosome reforation (ALR), restores the full complement of lysosomes in the cell. This evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation. 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 previously found that this death process was necrotic rather than apoptotic. In 2012, we carried out a study of the two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), that inhibit cell-cycle progression at the G2 phase of the cell cycle which led to cause necrotic cell death. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif kills the cell by inducing G2 arrest, we infected synchronized Jurkat human T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to mitosis phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was impaired. Finally, Vif-induced cell cycle arrest and cytotoxicity was correlated with proviral expression of Vif. we concluded that Vif impairs mitotic progression and causes fatal cell cycle disruption by interfering with Cdk1-CyclinB1 activation. In 2012, we also found that during autophagy, multiple lysosomes fuse with an autophagosome to form an autolysosome in which cytoplasmic components are sequestered and degraded by lysosomal hydrolases which releases the products into the cytosol via lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the "target of rapamycin" (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease similar to a sugar transporter. Spin mutants accumulate lysosomal carbohydrates and generate enlarged lysosomes in Drosophila and in human cells. We also demonstrated that spin is crucial for mTOR reactivation and lysosome reformation during prolonged starvation. Finally, we demonstrate that the sugar transporter activity of Spin is essential for ALR. These results provide a detailed molecular insight into how lysosome biogenesis proceeds during starvation and potentially during other autophagy-inducing conditions. We also conjecture that this mechanism may underlie normal lysosome biogenesis even in nutrient replete conditions.