Characterization of the Molecular Events of Autophagy

自噬分子事件的表征

• 批准号: 6463738
• 负责人: WILLIAM A DUNN
• 金额: $ 23.56万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-03 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6463738
• 关键词: autophagy; cell growth regulation; cell line; cytogenetics; electron microscopy; environmental stressor; enzyme activity; eukaryote; fluorescence microscopy; fungal genetics; fungal proteins; gene expression; immunoprecipitation; laboratory rabbit; membrane proteins; molecular genetics; neoplastic growth; polymerase chain reaction; protein binding; protein biosynthesis; protein kinase; protein protein interaction; protein structure function; site directed mutagenesis; vesicle /vacuole; yeast two hybrid system

DESCRIPTION: Eukaryotic cells adapt to environmental changes by altering their protein complements through synthesis and degradation. Cells that adapt poorly or improperly may either cease to exist (e.g., apoptosis) or become neoplastic (e.g., hepatoma). Cells adapt to low levels of amino acids by sequestering proteins and organelles for lysosomal degradation via a process called autophagy. The data from many laboratories suggest that autophagy is turned on during apoptosis and turned off during neoplastic growth implicating a role for autophagy in suppressing cancerous growth. Indeed, Beclin is a tumor suppressor that was originally shown to be required for autophagy in yeast. Our long-term goals are to characterize the molecular aspects of the regulation and mechanisms of cellular autophagy. We have characterized the degradation of peroxisomes and endogenous proteins by autophagy in the yeast Pichiapastoris under various environmental conditions. We have utilized this genetic model to identify 14 GSA genes that are required for glucose-induced selective autophagy. We have recently observed that during autophagy Gsa11 becomes associated with an organelle that is juxtaposed to the vacuole. In addition, this interaction requires the indirect or direct action of four additional GSA proteins including two protein kinases suggesting this event is highly regulated. Our results show that Gsa11 and its complex have a primary function in the sequestration of organelles for vacuole degradation. We propose that this organelle anchors at its surface a complex of proteins including Gsa11 that somehow organize the formation of the autophagic vacuole. In this application, we propose to examine the molecular and structural aspects of this organelle and the events required for the assembly of this complex in order to better understand its function. Our hypothesis is: Gsa11 must associate with a membrane-bound organelle prior to the sequestration events that occur during autophagy. We will utilize the versatility of our yeast model combined with a multidisciplinary approach of biochemical, cell biological, molecular biological, and genetic procedures to test our hypothesis. We will identify and characterize those GSA proteins that influence either directly or indirectly the formation of the Gsa11 complex. We will specifically evaluate the role of two protein kinases in the assembly of this complex. Finally, we will determine if the human homologue of Gsa11 is required for autophagy in a hepatoma cell line. The data obtained here will provide new insights into the events of sequestration of organelles for lysosomal degradation. In addition, with our new understanding of the molecular events of autophagy, we can begin to design clinical approaches by which to turn on autophagy and arrest neoplastic growth.

描述：真核细胞通过改变其自身结构来适应环境变化。 蛋白质通过合成和降解进行补充。适应能力差的细胞 或不当可能会停止存在（例如细胞凋亡）或变成肿瘤（例如肝癌）。细胞通过隔离来适应低水平的氨基酸 蛋白质和细胞器通过称为溶酶体降解的过程 自噬。许多实验室的数据表明自噬已开启 在细胞凋亡期间并在肿瘤生长期间关闭，这意味着 自噬抑制癌生长。事实上，Beclin 是一种肿瘤抑制剂 最初被证明是酵母自噬所必需的。我们的长期 目标是表征调节的分子方面和 细胞自噬机制。我们已经描述了退化的特征 酵母毕赤酵母中自噬作用的过氧化物酶体和内源蛋白 在各种环境条件下。我们利用这个遗传模型 鉴定出葡萄糖诱导选择性所需的 14 个 GSA 基因 自噬。我们最近观察到，在自噬过程中，Gsa11 变成 与与液泡并列的细胞器相关。此外， 这种相互作用需要四个额外的 GSA 的间接或直接作用 包括两种蛋白激酶的蛋白质表明该事件高度 受监管。我们的结果表明 Gsa11 及其复合物具有主要功能 隔离细胞器以进行液泡降解。我们建议 该细胞器在其表面锚定了包括 Gsa11 在内的蛋白质复合物 以某种方式组织自噬液泡的形成。在这个 应用程序，我们建议检查这个的分子和结构方面 细胞器以及组装该复合体所需的事件，以便 更好地理解它的功能。我们的假设是：Gsa11 必须在发生隔离事件之前与膜结合细胞器结合。 自噬。我们将利用酵母模型的多功能性与 生物化学、细胞生物学、分子生物学等多学科方法 生物学和遗传程序来检验我们的假设。我们将识别并 表征那些直接或间接影响的 GSA 蛋白 Gsa11复合物的形成。我们将具体评估 该复合物的组装中有两种蛋白激酶。最后，我们将确定 如果肝癌细胞中的自噬需要 Gsa11 的人类同源物 线。这里获得的数据将为事件提供新的见解 隔离细胞器以进行溶酶体降解。此外，与我们的 对自噬分子事件有了新的认识，我们可以开始设计 开启自噬并阻止肿瘤生长的临床方法。