Characterization of LC3-Associated Phagocytosis

描述（由申请人提供）：自噬是一种基本的细胞过程，其经典功能是清除长寿命蛋白质和回收细胞组分作为资源。在这个动态过程中，一个双膜隔离结构，称为自噬体，包围部分细胞质;这个新的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PI 3 K，VPS 34一起启动隔离膜或自噬载体的形成。细胞质材料周围的自噬体的延伸和最终闭合由两种泛素化样蛋白质缀合系统调节：ATG 12和LC 3-PE缀合途径。所得的ATG 5 -12-16复合物对于自噬体的扩张和/或弯曲以及LC 3的脂化形式LC 3-II的产生是必需的。ATG 5 -12-16复合物和LC 3-II都装饰自噬体的膜;然而，只有LC 3-II在与溶酶体融合期间保留。因此，认为LC 3-II对于自噬体靶向溶酶体细胞器是至关重要的。最近已经证明，TLR信号转导在吞噬过程中招募LC 3的吞噬体，在某种程度上依赖于ATG 5和ATG 7，并涉及Beclin-1和VPS 34协会。尽管使用了经典的自噬机制，但该过程在本文中被称为LC 3相关的吞噬作用（LC 3-associated phagocytosis）（LC 3-associated phagocytosis，LC 3-associated phagocytosis），缺乏使人联想到自噬体的双膜结构。总之，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着到吞噬体，导致吞噬体成熟加速和杀死摄入的病原体的能力增加。最近的一份报告描述了T和B细胞过度活跃，导致Tim 4缺陷小鼠淋巴细胞活化和自身免疫失调，Tim 4是吞噬细胞上的磷脂酰丝氨酸受体，有助于清除凋亡细胞。吞噬物质的加速清除中的作用表明，死亡细胞的有效清除可能需要与自噬机制的串扰。该提案的总体目标是阐明LC 3相关吞噬作用在垂死细胞摄取方面的作用和机制，以及表征其与传统自噬的差异。首先，我们将研究凋亡和坏死的死亡细胞诱导凋亡的能力。此外，我们将使用合成的抑制剂，siRNA和缺乏动物的原代细胞来研究启动信号下游的哪些分子是转录所需的。这些技术将有助于进一步定义自噬，就其与自噬的分歧而言。这些研究将包括检查ULK 1-ATG 13-FIP 200复合物、mTOR和Beclin 1-VPS 34-ATG 14 L-UVRAG复合物，这些复合物都是自噬反应的重要介质。此外，我们还将采用活细胞成像技术结合荧光标记分子来建立荧光蛋白所需的相关性，这是研究荧光蛋白的一个重要方法。这项研究将阐明所需的分子的噬菌体的特异性。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（或在这种情况下，自噬）的触发机制至关重要，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.