Characterization of the Molecular Events of Autophagy

描述：真核细胞通过改变它们的基因来适应环境变化。 蛋白质通过合成和降解来补充。适应能力差的细胞 或不适当地可能不复存在(例如，细胞凋亡)或变成肿瘤(例如，肝癌)。细胞通过隔离适应低水平的氨基酸 蛋白质和细胞器通过一个称为 自噬。来自许多实验室的数据表明，自噬已经开启 在细胞凋亡期间和在肿瘤生长期间被关闭，这涉及到 抑制癌细胞生长的自噬作用。事实上，贝克林是一名肿瘤抑制者 这最初被证明是在酵母中进行自噬所必需的。我们的长期合作 目标是描述调控的分子方面，并 细胞自噬的机制。我们已经描述了退化的特征 毕赤酵母中自噬产生的过氧化物体和内源蛋白 在各种环境条件下。我们已经利用这种遗传模型 确定葡萄糖诱导的选择性所需的14个GSA基因 自噬。我们最近观察到，在自噬过程中，Gsa11成为 与液泡并列的细胞器相关的。此外, 这种互动需要另外四个GSA的间接或直接行动 包括两个蛋白激酶在内的蛋白质表明这一事件高度 受监管的。我们的结果表明，Gsa11及其复合体具有主要功能 在液泡降解的细胞器的隔离中。我们建议 这种细胞器在其表面锚定了一种包括Gsa11在内的蛋白质复合体 以某种方式组织了自噬空泡的形成。在这 应用，我们建议研究它的分子和结构方面 细胞器和组装这个复合体所需的事件，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的细胞器联系在一起 自噬。我们将利用我们的酵母模型的多功能性，结合 生物化学、细胞生物学、分子生物学的多学科研究方法 生物学和遗传学程序来验证我们的假设。我们将确定和 描述那些直接或间接影响GSA的蛋白质 Gsa11复合体的形成。我们将具体评估 在这个复合体的组装中有两个蛋白激酶。最后，我们将确定 如果需要Gsa11的人类同源物在肝癌细胞中进行自噬 排队。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，通过我们的 对自噬分子事件的新认识，我们可以开始设计 开启自噬和阻止肿瘤生长的临床方法。

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.