Characterization of the Molecular Events of Autophagy

自噬分子事件的表征

• 批准号: 6794642
• 负责人: WILLIAM A DUNN
• 金额: $ 23.52万
• 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/6794642
• 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：真核细胞通过改变它们的 通过合成和降解补充蛋白质。适应能力差的细胞 或者不适当地可能不再存在（例如，凋亡）或成为肿瘤（例如，肝癌）。细胞通过螯合来适应低水平的氨基酸 蛋白质和细胞器的溶酶体降解通过一个过程， 自噬许多实验室的数据表明， 在细胞凋亡过程中和肿瘤生长过程中关闭， 自噬抑制癌细胞生长。事实上，Beclin是一种肿瘤抑制因子， 最初被证明是酵母中自噬所必需的。我们的长期 目标是表征调控的分子方面， 细胞自噬的机制我们已经描述了 毕赤酵母中过氧化物酶体和内源蛋白的自噬作用 在各种环境条件下。我们利用这种遗传模型， 鉴定了葡萄糖诱导的选择性表达所需的14个GSA基因， 自噬我们最近观察到，在自噬过程中， 与液泡并列的细胞器有关。此外，本发明还提供了一种方法， 这种相互作用需要另外四个全球服务机构的间接或直接行动 包括两种蛋白激酶的蛋白质，表明该事件是高度 监管.我们的研究结果表明，Gsa 11及其复合物具有主要功能， 在细胞器的隔离中用于液泡降解。我们建议 这种细胞器在其表面固定了一种蛋白质复合物， 以某种方式组织自噬泡的形成。在这 应用，我们建议检查分子和结构方面的这一点， 细胞器和组装这个复合体所需的事件，以 更好地理解它的功能。我们的假设是：Gsa 11必须与一个膜结合的细胞器之前发生的隔离事件， 自噬我们将利用酵母模型的多功能性， 生物化学、细胞生物学、分子生物学 生物学和遗传学的方法来验证我们的假设我们将查明和 描述那些直接或间接影响 Gsa 11复合物的形成。我们将具体评估以下方面的作用： 两种蛋白激酶参与了这个复合物的组装。最后，我们将确定 如果Gsa 11的人类同源物是肝癌细胞中自噬所必需的， 线这里获得的数据将为我们提供对 用于溶酶体降解的细胞器的隔离。此外，与我们的 对自噬的分子事件有了新的理解，我们就可以开始设计 临床方法，通过这些方法来开启自噬并阻止肿瘤生长。