Characterization of LC3-Associated Phagocytosis

描述（由申请人提供）：自噬是一个基本的细胞过程，其经典功能是清除长寿命蛋白质和循环利用细胞成分作为资源。在这一动态过程中，称为自噬体的双膜隔离结构包围着部分细胞质；这个新细胞器的内容物随后被递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细胞的形成。自噬细胞在细胞质物质周围的延伸和最终闭合是由两种泛素化样蛋白质偶联系统调节的：ATG12和LC3-PE偶联途径。由此产生的ATG5-12-16复合物对于自噬体的扩张和/或弯曲以及脂化版本LC3， LC3- ii的产生是必需的。ATG5-12-16复合体和LC3-II都修饰自噬体的膜；然而，只有LC3-II在与溶酶体融合过程中保留下来。因此，我们认为LC3-II对于自噬体靶向溶酶体细胞器至关重要。最近有研究表明，在吞噬过程中，TLR信号以依赖于ATG5和ATG7的方式将LC3招募到吞噬体，并涉及Beclin-1和VPS34的关联。尽管使用了经典的自噬机制，但这一过程，在这里被称为lc3相关吞噬（LAP），缺乏让人想起自噬体的双膜结构。综上所述，这些数据表明了吞噬和自噬途径的趋同，其中经典的自噬机制附着在吞噬体上，导致吞噬体成熟加速，杀死摄入病原体的能力增强。最近的一份报告描述了T和B细胞过度活跃，导致淋巴细胞激活和自身免疫失调，在缺乏Tim4的小鼠中，Tim4是吞噬细胞上的磷脂酰丝氨酸受体，促进凋亡细胞的清除。LAP在加速吞噬物质清除中的作用表明，死亡细胞的有效清除可能需要与自噬机制进行串扰。本研究的总体目标是阐明lc3相关吞噬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（凋亡细胞和坏死细胞）诱导LAP的能力。此外，我们将使用合成抑制剂、siRNA和来自缺陷动物的原代细胞来研究LAP需要启动信号下游的哪些分子。这些技术将有助于进一步定义LAP，因为它与自噬的差异。这些研究将包括检查ULK1-ATG13-FIP200复合体、mTOR和Beclin1-VPS34- ATG14L-UVRAG复合体，这些都是自噬反应的重要介质。此外，我们将研究LAP的一个重要方法是使用活细胞成像与荧光标记分子相结合，以建立LAP所需的关联。本研究将阐明LAP对吞噬体规范所需的分子。新出现的数据表明自噬是癌症的一个重要方面；然而，这种作用似乎是矛盾的，因为自噬已被报道具有细胞保护和抗肿瘤作用。事实上，进一步了解自噬（在本例中为LAP）触发的机制将是至关重要的，从而为研究人员提供新的治疗靶点。

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.