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: 8336105
• 负责人: michael j lenardo
• 金额: $ 59.95万
• 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/8336105
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Amino Acids; Apoptosis; Apoptotic; Autophagocytosis; Autophagosome; Binding; Biochemical; Biogenesis; Biological; CD4 Positive T Lymphocytes; CD95 Antigens; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Death; Cell Surface Receptors; Cell Survival; Cells; Cellular Morphology; Cessation of life; Complement; Complex; Cyclin-Dependent Kinases; Cyclins; Cytoplasmic Organelle; Cytoplasmic Protein; Degenerative Disorder; Digestion; Disease; Drosophila spinster protein; Equilibrium; Exhibits; G2 Phase; Genes; Genetic; Genetic Programming; Golgi Apparatus; HIV; HIV-1; Homeostasis; Homologous Gene; Host Defense; Human; Immune; Immune response; Infection; Infectious Agent; Investigation; Lead; Lymphocyte; Lysosomes; Mammalian Cell; Membrane; Metabolism; Mitosis; Mitotic; Molecular; Molecular Biology; Molecular Genetics; Morbidity - disease rate; Morphology; Natural regeneration; Necrosis; Normal Cell; Nutrient; Open Reading Frames; Organ; Outcome; Pathway interactions; Peptide Hydrolases; Phosphotransferases; Play; Process; Protein Isoforms; Proteins; Reactive Oxygen Species; Receptor Signaling; Regulation; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Signal Transduction; Starvation; T-Lymphocyte; TNFRSF1A gene; TNFRSF6 gene; Tumor Necrosis Factor Receptor; Vesicle; Viral; Viral Proteins; Virus; Virus Diseases; Work; Yeasts; alpha helix; caspase-8; catalase; cell growth regulation; cell injury; cell type; chemotherapeutic agent; cytotoxic; detection of nutrient; human FRAP1 protein; insight; interest; late endosome; mathematical model; mortality; neoplastic cell; palliative; pathogen; preference; programs; protein function; receptor; response; tissue culture; trafficking; transcription factor; vpr Gene Products

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 reformation at the end of autophagy. 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 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.We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, of SARS-CoV and found that ORF 3a accounted for cell death caused by this virus. In addition, ORF 3a causes SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles. The overarching conclusion that we can draw from these studies is that cell death can be programmed in many different ways in the cell depending on the cell-type and conditions. Various cell biological mechanisms can participate which means that the genetic control varies depending on the specific mechanism of death. By understanding the molecular mechanisms of death, we hope to gain better insight into normal cellular homeostasis as well as degenerative disease processes.

内部死亡计划在许多疾病中起着重要作用。 致病作用可能是由于细胞死亡效率低下或不适当或过度死亡所致，例如在SARS期间由人类免疫缺陷病毒（HIV）或SAR-COV病毒引起的。在这个项目中，我们正在采用多方面的方法来研究淋巴细胞以及其他细胞类型中凋亡和非凋亡死亡程序的分子机制。我们研究的主要重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，例如TNFR1和CD95/FAS/APO-1。 两种受体在刺激细胞的凋亡和非凋亡死亡中都起着重要作用，主要是在免疫过程中。关于这些替代死亡途径如何涉及受体信号传导，知之甚少。 有趣的是，两种受体除死亡以外的作用，例如转录因子的诱导。我们正在尝试了解这些受体如何刺激偏爱其他细胞结局的细胞内机械。我们致力于了解调节死亡计划的蛋白酶的激活。我们表征了两个从TNFR1和FAS受体中散发出来的死亡程序，一种是caspase-8依赖性的，具有凋亡形态，另一个是caspase-8独立的，涉及坏死。有趣的是，只有在抑制caspase-8时才观察到后一种死亡计划。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，表现出特定的细胞质双膜结构，称为自噬。 尽管最初引起争议，但现在有几个实验室表明，这种死亡形式对于化学治疗剂肿瘤细胞的灭亡尤为重要。现在，我们已经表明，自噬死亡程序的机制是过氧化氢酶的选择性降解，这导致反应性氧物种的明显过度累积导致细胞损伤和死亡。此外，我们专注于在这一死亡过程中起关键作用的基因。 我们发现，果蝇杂质蛋白（称为Hspin）的人类同源物对于自噬细胞死亡至关重要。 我们已经研究了该蛋白质的生化功能，发现它对于适当的溶酶体生物发生和囊泡运输很重要。特别是，它在自噬结束时在溶酶体改革中起着至关重要的作用。 自噬是一个从人到酵母的进化保守的过程，通过该过程，细胞质蛋白和细胞器被分解代谢，但是当细胞在自噬细胞死亡和生存之间选择自动噬时的结果时，对结果知之甚少。在饥饿期间，蛋白质TOR（雷帕霉素的靶标）是一种控制细胞代谢的营养反应性激酶，被关闭并激活自噬。双膜自噬体隔离细胞内成分，然后与溶酶体融合以形成自体染色体，从而将其含量分解成再生营养素。我们对自噬的理解是，它在自溶解体内终止了货物降解，但是自噬如何由营养素控制，随后的自溶性命运是未知的。我们发现在自噬开始期间抑制哺乳动物细胞中的mTOR信号传导，但在延长的饥饿期间被重新激活。 MTOR的重新激活取决于自溶剂体产物的降解和养分的释放。 MTOR活性依次终止自噬并刺激令人印象深刻的原始溶质体小管和囊泡，这些小管和囊泡从自溶液中挤出并最终成熟成功能性溶酶体。 这个过程，我们将自噬溶酶体炼油（ALR）称为此过程，它恢复了细胞中溶酶体的完整补体。自噬中，这种进化保守的循环控制饥饿期间的营养感应和溶酶体体内平衡。 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后艾滋病的临时作用是该病毒引起的T淋巴细胞死亡。我们发现，这种死亡过程是坏死的，而不是凋亡，现在已经鉴定出参与此过程的两个病毒基因产品VIF和VPR。我们发现，VPR通过与在细胞周期进程中起作用的细胞蛋白结合来改变细胞周期并促进死亡。 为了严格研究这一过程，我们构建了一个数学模型，以分析HIV感染期间组织培养的细胞死亡。值得注意的是，这两种细胞毒性基因产物引起的都表明在G2和M相边界处的循环停滞。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发生。 HIV VPR蛋白是一种小蛋白（100个氨基酸），没有明显的结构结构域或三个α螺旋以外的酶基序。 我们已经确定，VPR促进了有丝分裂调节剂（例如Cyclinb和Cdk1）和14-3-3蛋白的THETA同工型之间显然流产的复合物的形成，该蛋白抑制了G2相中的细胞周期。 该复合物似乎是通过VPR蛋白的第三个螺旋上的特定疏水贴剂成核的。我们还研究了VIF如何引起细胞周期停滞，发现它是VPR引起的一种独特的机制。 我们发现VIF可以改变细胞周期蛋白和细胞周期蛋白依赖性激酶的核质质定位，从而导致正常细胞周期进程的破坏。 我们继续探索HIV-1如何改变细胞机制以引起CD4 T细胞的灭亡。我们研究了SARS-COV的最大辅助蛋白ORF 3A蛋白的功能，并发现ORF 3A占该病毒引起的细胞死亡。另外，ORF 3A导致SARS-COV诱导的高尔基片段化，并且3A蛋白会积聚并定位在含有晚期内体标记的囊泡上。这些结果在SARS-COV诱导的细胞死亡，高尔基体碎片和细胞内囊泡的积累中确定了ORF 3A的重要作用。 我们可以从这些研究中得出的总体结论是，可以根据细胞类型和条件以许多不同的方式对细胞死亡进行编程。 各种细胞生物学机制可以参与，这意味着遗传控制取决于死亡的特定机制。 通过了解死亡的分子机制，我们希望能够更好地了解正常的细胞稳态以及退行性疾病过程。