多細胞生物吞噬的調節機制

張宏

摘 要：該研究以秀麗線蟲為模型，以細胞自吞噬為中心，用遺傳學、細胞生物學、生化、蛋白質組學等多種手段全面系統地鑒定多細胞生物中參與自吞噬過程的所有組成基因，研究自吞噬的調節機制。同時，還著重研究自吞噬與另外兩個基本的生命過程——細胞凋亡和個體衰老之間的相互關系和調節作用。該研究共分為4部分：第一，建立了以秀麗線蟲為研究細胞自噬的多細胞生物模型，通過遺傳學手段篩選并克隆新的細胞自噬分子，闡明其作用的分子、細胞及遺傳調控機制，揭示這些新分子在個體發育過程中的功能，并為哺乳動物細胞中的同源基因的研究提供線索。第二，研究了凋亡細胞清除的多個環節，發現了在凋亡細胞清除的不同階段發揮作用的新調控因子并闡明了其作用機制。成果包括：橋聯分子TTR-52介導凋亡細胞識別及吞噬的調控機制；TTR-52、CED-7及脂結合/轉運蛋白NRF-5協同調控PS，然后吞噬細胞表面的呈現；肌管素磷酸酶MTM-1負調控凋亡細胞吞噬。第三，通過比較長壽daf-2突變體線蟲和野生型線蟲的線粒體，包括形態、生理活性特征、線粒體DNA的拷貝數和線粒體蛋白質組的差異，找出daf-2突變體長壽的原因，探索在多細胞動物中細胞自體吞噬與衰老之間的關系，以及胰島素信號調節線粒體和細胞自吞噬的作用機制。第四，開發和完善了各項基于生物質譜的蛋白質組學技術，利用這些生物質譜技術對哺乳動物細胞自吞噬的起始進行了深入的研究和探討。同時，還將開發的生物質譜技術廣泛應用到各個重要的生物學領域中，推動了生物學研究領域的發展。

關鍵詞：秀麗線蟲 細胞自噬 細胞凋亡 衰老 質譜

Mechanism of Autophagy in Multicellular Organism

Zhang Hong

（National Institute of Biological Sciences， Beijing）

Abstract：We used C. elegans as a multicellular genetic model system to investigate molecular mechanisms of autophagy， apoptosis and aging. The project aimed to address the following questions： the molecular machinery of the evolutionarily conserved autophagy-lysosome pathway， the phagocytosis pathway and also how these processes affect the aging process during animal development. The research includes the following aspects： First， we established C. elegans as a multicellular genetic model to delineate the autophagic machinery by demonstrating that a variety of protein aggregates are selectively removed by autophagy during embryogenesis. We also investigated the physiological function of these metazoan-specific autophagy genes in mice. Second， we studied the phagocytic removal of apoptotic cells， which is an integral part of the cell death program and an important event in tissue remodeling， suppression of inflammation and regulation of the immune response. We have identified six novel regulators and revealed mechanisms through which they regulate various aspects of cell corpse removal. Among these newly identified regulators， TTR-52 and NRF-5 are extracellular lipid-binding/transfer proteins that mediate recognition of cell corpses. Third， we investigated the role of mitochondria in aging process. Using metabolic labeling with the stable heavy isotope 15N and quantitative mass spectrometry （MS）， we find that proteins up-regulated in daf-2 are highly enriched for those functioning in fatty acid metabolism， glyoxylate cycle， amino acid metabolism， or ROS metabolism. Fourth， we set up a high resolution biological mass spectrometer. According to the research directions proposed by the grant， we developed a variety of mass spectrometry based proteomics techniques. Through biological mass spectrometry， we identified multiple phosphorylation sites on proteins that help initiate autophagy in mammalian cells. Besides the field of autophagy， we also applied the biological mass spectrometric techniques in a variety of important biological fields including necrosis and aging， contributing greatly to the advancement of these fields. Our studies help us to understand the molecular autophagic pathway and also the physiological function in multicellular organisms and also how the autophagy-lysosomal pathway interacts with the endosomal-lysosomal pathway.

Key Words：C. elegans； Autophagy； Apoptosis； Aging； Mass

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