MECHANISM OF AUTOPHAGY

自噬是细胞内细胞器被 在溶酶体空泡系统内被隔离和降解。 它是生物降解的重要途径 内源性蛋白质，不仅在分化，变态 和衰老之间的关系，以及正常的蛋白质周转。 详细事件 自噬的调节和机制仍然未知 并形成我长期研究目标的基础。 在这 项目，我已经提议在大鼠肝脏中研究以下事件： 自噬泡（AV）的形成和成熟， 溶酶体 由于空泡的形成和随后的融合 与溶酶体是膜事件，我的重点将是在 AV膜的表征。 空泡膜 似乎来自于预先存在的细胞质膜。 使用细胞器特异性抗体结合 免疫过氧化物酶细胞化学，我已经表明，这些 液泡不是由高尔基体或质膜形成的 成分，但它们可能来自RER， 溶酶体 我现在要准备单特异性抗体 粗面内质网和溶酶体的膜蛋白来证明我最初的 更详细地发现和检查自噬的途径。 使用胰高血糖素促进空泡形成和低血糖 温度，以抑制进入溶酶体水解酶，我有 建立的条件导致新的 形成了AV。 我现在将使用单特异性抗体（即， 免疫金定位在电子显微镜水平）， 鉴定特异性膜抗原并确定是否所有AV都 由RER形成或者如果存在次级源（例如， 溶酶体）。 我的结果显示 AV由粗面内质网的光滑膜区域形成， 也将确定是否有一个损失之间的关系， 膜结合的多聚核糖体和自噬的发生。 我会 首先测量肝脏中膜结合核糖体的水平 在富含营养的条件下灌注（自噬抑制）， 营养耗尽（自噬刺激）条件，然后 比较稳定条件下的自噬反应， （环己酰亚胺）或使（组氨醇）多核糖体不稳定。 最后， 我将研究细胞损伤对肝脏自噬的影响， 为了确定自噬反应是否类似于 在生理刺激过程中的特征。 调查 可能促进这种反应的替代条件将 在评价促进人权的机制方面变得重要， 自噬的调节。

Autophagy is a process by which intracellular organelles are sequestered and degraded within the lysosomal vacuolar system. It is a biologically important pathway for the degradation of endogenous proteins, not only in differentiation, metamorphosis and aging but also in normal protein turnover. The detailed events in the regulation and mechanism of autophagy are still unknown and form the basis of my long range research goals. In this project, I have proposed to study in rat liver the events of autophagic vacuole (AV) formation and maturation into secondary lysosomes. Since formation of the vacuoles and subsequent fusion with lysosomes are membrane events, my focus will be on the characterization of the AV membrane. The vacuolar membranes appear to be derived from pre-existing cytoplasmic membranes. Using organelle-specific antibodies combined with immunoperoxidase cytochemistry, I have shown that these vacuoles are not formed from Golgi or plasma membrane constituents but that they are probably derived from RER and lysosomes. I will now prepare monospecific antibodies to specific membrane proteins of the RER and lysosome to prove my initial finding and examine in more detail the pathway of autophagy. Using glucagon to promote vacuole formation and low temperature to inhibit entry of lysosomal hydrolases, I have established conditions which result in the accumulation of newly formed AV's. I will now use monospecific antibodies (i.e., immunogold localization at the electron microscope level) to identify specific membrane antigens and determine if all AV's are formed from the RER or if there is a secondary source (e.g., lysosome) of these vacuoles. With my results indicating that the AV's are formed from smooth membrane regions of the RER, I will also determine if there is a relationship between the loss of membrane-bound polyribosomes and the onset of autophgy. I will first measure the levels of membrane-bound ribosomes in livers perfused under nutrient-enriched (autophagy inhibited) and nutrient-depleted (autophagy stimulated) conditions and then compare the autophagic response under conditons that stabilize (cycloheximide) or destabilize (histidinol) polyribosomes. Finally, I will examine the effects of cell injury on autophagy in the liver, in an effort to determine if the autophagic response is similar to that characterized during physiological stimuli. A survey of alternative conditions which may promote this response will become important in an evaluation of the mechanisms for the regulation of autophagy.