Characterization of LC3-Associated Phagocytosis

描述(申请人提供)：自噬是一个基本的细胞过程，其经典功能是清除长寿命的蛋白质和回收细胞成分的资源。在这一动态过程中，一个被称为自噬小体的双层膜隔离结构包围着细胞质的部分；这个新细胞器的内容随后被输送到溶酶体进行降解。简而言之，ATG蛋白与Beclin-1和III类PI3K，Vps34一起启动隔离膜或自噬载体的形成。自噬细胞在细胞质物质周围的伸长和最终闭合受两个泛素化类蛋白质结合系统的调节：ATG12和Lc3-PE结合途径。由此产生的ATG5-12-16复合体对于自噬小体的扩张和/或曲率以及脂化版本Lc3，Lc3-II的产生是必需的。ATG5-12-16复合体和LC3-II都装饰着自噬体膜；然而，只有LC3-II在与溶酶体融合的过程中保留下来。因此，LC3-II被认为是将自噬小体靶向溶酶体细胞器的关键。最近的研究表明，吞噬过程中的TLR信号以依赖于ATG5和ATG7的方式将LC3募集到吞噬小体中，并涉及Beclin-1和Vps34的关联。尽管使用了经典的自噬机制，但这个过程，在这里被称为LC3相关吞噬作用(LAP)，缺乏使人想起自噬小体的双层膜结构。综上所述，这些数据表明吞噬和自噬途径的融合，其中经典的自噬机制被附在吞噬小体上，导致吞噬小体加速成熟，并增强杀死吞噬病原体的能力。最近的一份报告描述了缺乏Tim4的小鼠的T和B细胞过度活动，导致淋巴细胞激活和自身免疫失调。Tim4是吞噬细胞上的一种磷脂酰丝氨酸受体，有助于清除凋亡细胞。LAP在快速清除吞噬物质方面的作用表明，也许死亡细胞的有效清除可能需要与自噬机制的串扰。这项建议的总体目标是阐明LC3相关的吞噬作用在死亡细胞摄取方面的作用和机制，并表征其与传统自噬的差异。首先，我们将研究死亡细胞，包括凋亡性和坏死性，诱发LAP的能力。此外，我们将使用合成抑制剂、siRNA和缺陷动物的原代细胞来研究LAP需要哪些启动信号下游的分子。根据LAP与自噬的区别，这些技术将有助于进一步定义LAP。这些研究将包括对ULK1-ATG13-FIP200复合体、mTOR和Beclin1-Vps34-ATG14L-UVRAG复合体的检测，这些都是自噬反应的重要中介。此外，我们将使用活细胞成像和荧光标记的分子来建立LAP所需的关联，这将是研究LAP的一个重要方法。这项研究将阐明吞噬小体LAP规范所需的分子。新出现的数据表明，自噬是癌症的一个重要方面；然而，这种作用似乎是自相矛盾的，因为据报道，自噬具有细胞保护和抗肿瘤作用。事实上，进一步了解自噬或在这种情况下引发LAP的机制将是至关重要的，从而为研究人员提供新的治疗目标。

DESCRIPTION (provided by applicant): Autophagy is a fundamental cellular process whose classical function is the clearance of long-lived proteins and the recycling of cellular components for resources. During this dynamic process, a double membrane isolation structure, termed the autophagosome, surrounds portions of the cytoplasm; the contents of this new organelle are subsequently delivered to the lysosome for degradation. In short, ATG proteins, with Beclin-1 and the class III PI3K, VPS34, initiate the formation of the isolation membrane or autophagophore. The elongation and ultimate closure of the autophagophore around the cytoplasmic material is regulated by two ubiquitylation- like, protein conjugation systems: the ATG12 and LC3-PE conjugation pathways. The resulting ATG5-12-16 complex is necessary for the expansion and/or curvature of the autophagosome, as well as the generation of the lipidated version of LC3, LC3-II. Both the ATG5-12-16 complex and LC3-II decorate the membranes of the autophagosome; however, it is only LC3-II that remains during its fusion with the lysosome. It is therefore believed that LC3-II is critical for the targeting of autophagosomes to lysosomal organelles. It has recently been demonstrated that TLR signaling during phagocytosis recruits LC3 to the phagosome, in a manner dependent on ATG5 and ATG7 and involving Beclin-1 and VPS34 association. Despite the use of classical autophagy machinery, this process, herein referred to as LC3-associated phagocytosis (LAP), lacks the double membrane structure reminiscent of the autophagosome. Taken together, this data suggest a convergence of the phagocytic and autophagic pathways, where classical autophagy machinery is annexed to the phagosome resulting in accelerated phagosome maturation and increased ability to kill ingested pathogens. A recent report has described T and B cell hyperactivity, leading to dysregulated lymphocyte activation and autoimmunity, in mice deficient for Tim4, a phosphatidylserine receptor on phagocytes that facilitates the clearance of apoptotic cells. The role of LAP in the expedited clearance of phagocytosed material indicates that perhaps efficient clearance of dying cells could require crosstalk with the autophagic machinery. The overall goal of this proposal is to elucidate the role and mechanisms of LC3-associated phagocytosis in terms of uptake of dying cells, as well as characterize its divergence from traditional autophagy. Firstly, we will investigate the ability of dying cells, both apoptotic and necrotic, to induce LAP. Furthermore, we will use synthetic inhibitors, siRNA, and primary cells from deficient animals to investigate which molecules downstream of the initiating signal are required for LAP. These techniques will aid to further define LAP, in terms of its divergence from autophagy. These studies will include examination of the ULK1-ATG13-FIP200 complex, mTOR, and the Beclin1-VPS34- ATG14L-UVRAG complex, all important mediators of the autophagic response. In addition, we will An important approach to studying LAP will be the use of live cell imaging in conjunction with fluorescently-labeled molecules to establish the associations required for LAP. This study will elucidate the molecules required for LAP specification to the phagosome. Emergent data indicates that autophagy is an important aspect of cancer; this role, however, seems paradoxical, as autophagy has been reported to have both cytoprotective and anti- tumor effects. Indeed, it will be critical to further understand the mechanisms by which autophagy, or in this case, LAP, can be triggered, thus providing researchers with novel therapeutic targets.