Molecular Pathways of Programmed C ell Death And Viral Cytopathicity

程序性细胞死亡和病毒细胞病变的分子途径

• 批准号: 8555809
• 负责人: michael j lenardo
• 金额: $ 30.55万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555809
• 关键词: Acquired Immunodeficiency Syndrome; Apoptosis; Apoptotic; Autophagocytosis; Autophagosome; Biogenesis; Carbohydrates; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Death; Cell Surface Receptors; Cell Survival; Cells; Cellular Morphology; Cessation of life; Characteristics; Chief Cell; Complement; Cytoplasmic Organelle; Cytoplasmic Protein; Cytosol; Digestion; Disease; Drosophila genus; Equilibrium; Exhibits; G2 Phase; Genes; Genetic Programming; HIV; HIV-1; Homeostasis; Host Defense; Human; Hydrolase; Immune; Immune response; Infection; Infectious Agent; Investigation; Lead; Lymphocyte; Lysosomes; Mammalian Cell; Membrane; Metabolism; Mitosis; Mitotic; Molecular; Molecular Biology; Morbidity - disease rate; Morphology; Natural regeneration; Necrosis; Nuclear Translocation; Nutrient; Organ; Outcome; Pathway interactions; Phase Transition; Phosphotransferases; Play; Process; Protein Dephosphorylation; Reactive Oxygen Species; Receptor Signaling; Regulation; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Signal Transduction; Sirolimus; Starvation; T-Lymphocyte; TNFRSF1A gene; TNFRSF6 gene; Tumor Necrosis Factor Receptor; Vesicle; Viral; Viral Proteins; Virus; Virus Diseases; Work; Yeasts; caspase-8; catalase; cell growth regulation; cell injury; cell killing; cell type; chemotherapeutic agent; cytotoxicity; detection of nutrient; human FRAP1 protein; inorganic phosphate; insight; interest; mortality; mutant; neoplastic cell; palliative; pathogen; permease; preference; programs; receptor; response; sugar; transcription factor

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.

内部死亡程序在许多疾病中发挥着重要作用。 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。关于这些替代性死亡途径如何被带入受体信号传导，人们知之甚少。 有趣的是，这两种受体除了死亡之外还可以产生作用，例如诱导转录因子。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。我们已经发现，在非淋巴细胞中抑制半胱天冬酶-8可以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双膜结构。 尽管最初存在争议，但几个实验室现在已经表明，这种形式的死亡对化疗药物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。 自噬是一个从人类到酵母的进化保守过程，细胞质蛋白和细胞器被分解代谢，但对自噬结束时细胞在自噬细胞死亡和存活之间选择的结果知之甚少。在饥饿期间，蛋白质TOR（雷帕霉素的靶蛋白），一种控制细胞代谢的营养响应激酶，被关闭，自噬被激活。双膜自噬体将细胞内的成分隔离，然后与溶酶体融合形成自溶体，自溶体将其内容物分解代谢以再生营养。我们目前对自噬的理解是，它终止于自溶酶体内的货物降解，但自噬如何受营养素控制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信号在自噬启动期间被抑制，但在长期饥饿期间被重新激活。mTOR的再活化依赖于自体溶酶体产物的降解和营养素的释放。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，其从自体溶酶体中挤出并最终成熟为功能性溶酶体。 这个过程，我们称之为自噬溶酶体再形成（ALR），恢复了细胞中溶酶体的全部补充。这种进化上保守的自噬循环控制着饥饿期间的营养感测和溶酶体体内平衡。 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。我们以前发现，这种死亡过程是坏死而不是凋亡。2012年，我们对人类免疫缺陷病毒1型（HIV-1）中的两种主要细胞病变因子进行了研究，即辅助蛋白病毒感染因子（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，导致坏死性细胞死亡。虽然Vpr诱导的阻断和相关的T细胞死亡已经得到了很好的研究，G2期阻滞的Vif的分子机制仍然不确定。为了阐明Vif如何通过诱导G2期阻滞杀死细胞，我们感染了同步化的Jurkat人T细胞，并研究了Vif对Cdk 1和CyclinB 1激活的影响，Cdk 1和CyclinB 1是G2期到有丝分裂期转变的主要细胞周期因子。我们发现，特征去磷酸化的抑制磷酸对Cdk 1没有发生在感染的细胞表达Vif。此外，Cdk 1和CyclinB 1的核转位也受到了抑制。最后，Vif诱导的细胞周期阻滞和细胞毒性与Vif的前病毒表达相关。我们的结论是Vif通过干扰Cdk 1-CyclinB 1的活化而损害有丝分裂进程并导致致命的细胞周期破坏。 在2012年，我们还发现在自噬期间，多个溶酶体与自噬体融合以形成自溶酶体，其中细胞质组分被溶酶体水解酶隔离和降解，所述溶酶体水解酶通过溶酶体外排渗透酶将产物释放到胞质溶胶中。饥饿诱导的自噬后，通过自噬性溶酶体重组（ALR）恢复溶酶体稳态，需要激活“雷帕霉素靶点”（TOR）激酶。Spinster（Spin）编码一种推定的溶酶体外排通透酶，类似于糖转运蛋白。自旋突变体积累溶酶体碳水化合物，并在果蝇和人类细胞中产生扩大的溶酶体。我们还证明，自旋是至关重要的mTOR重新激活和溶酶体改造在长期饥饿。最后，我们证明了Spin的糖转运活性对ALR是必不可少的。这些结果提供了一个详细的分子洞察溶酶体生物合成如何进行饥饿期间，并可能在其他自噬诱导条件。我们还推测，这种机制可能是正常的溶酶体生物合成的基础，即使在营养丰富的条件下。