Regulation of lytic granule exocytosis in Natural Killer (NK) Cells

自然杀伤 (NK) 细胞中裂解颗粒胞吐作用的调节

基本信息

批准号：
8946386
负责人：
JOHN COLIGAN
金额：
$ 20.78万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8946386
关键词：
Abnormal Cell Adaptor Signaling Protein Address Affect Apoptosis Autophagocytosis Binding Biogenesis Caspase Cell Degranulation Cell Line Cell membrane Cell surface Cells Cellular biology Cessation of life Characteristics Complex Cytoplasm Cytoplasmic Granules Cytoskeleton Defect Disease Excision Exocytosis Fibroblasts Glycogen Storage Disease Type IIb Goals Golgi Apparatus Granzyme Griscelli Syndrome Hemophagocytic Lymphohistiocytoses Human Impairment Knowledge Lead Lymphocyte Subset Lysosomes LytA enzyme Lytic Membrane Membrane Fusion Membrane Proteins Movement Mus Mutation Natural Killer Cells Pathway interactions Phagosomes Phenotype Protein Import Proteins RNA Interference Regulation Role Site Sorting - Cell Movement Surface Syndrome Tissues Transport Vesicles Vacuole cell motility chediak-higashi syndrome cytokine cytotoxic cytotoxicity familial hemophagocytic lymphohistiocytosis granzyme B immunological synapse killings microbial pathogen perforin trafficking tumor

项目摘要

Natural killer (NK) cells comprise a subset of lymphocytes involved in protection against microbial pathogens and tumors. Because of their cytotoxic capacity, NK cells are able to directly eliminate abnormal cells. Lytic granules of NK cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Defects in lytic granule secretion are associated with serious diseases, such as hemophagocytic lymphohistiocytosis and Chediak-Higashi, Hermansky-Pudlak, or Griscelli syndromes. Knowledge about the proteins involved in regulation of lytic granule release is very limited, and the granule-specific protein machinery involved in NK-cell exocytosis is poorly understood. Lytic granules have the characteristics of lysosomes and are referred to as secretory lysosomes. There are many proteins residing in lysosomal membranes that function in acidification of the lysosomal lumen, protein import, membrane fusion, and transport of degradation products to the cytoplasm. Surprisingly, the role of the most abundant proteins, lysosome-associated membrane protein (LAMP) 1 and LAMP2 (CD107a and CD107b, respectively), is ill-defined. In humans, mutations in LAMP2 cause Danon disease, believed to arise from aberrant autophagy. LAMP2-deficient mice also accumulate autophagic vacuoles in several tissues. LAMP1-deficient mice have an almost normal phenotype but have elevated levels of LAMP2, suggesting that LAMP2 can compensate for the loss of LAMP1. Recent studies using LAMP-depleted fibroblasts demonstrate that both proteins are required for proper lysosomal transport and efficient (auto)phagosome-lysosome fusion. LAMP1 is also widely used as a marker for the identification of NK-cell degranulation, as it appears on the cell surface following the fusion of lysosomes with the plasma membrane, but the role of LAMP1 in NK-cell biology is completely unknown. Since LAMP1 is one of the most abundant proteins in the lysosomal membrane and NK cells use their lysosomes to induce the death of target cells, we sought to determine the importance of LAMP1 in NK-cell cytolytic activity. Therefore, this project emphasis has been to understand the role of lysosomal membrane protein 1, LAMP1, in regulation of human NK cell cytotoxicity. To address this issue, RNA interference (RNAi) was used to disrupt LAMP1 expression in human NK cell lines, as well as primary NK cells, and the effects of LAMP1 silencing on lytic granule composition, movement, exocytosis and NK cell cytotoxic potential were investigated. We have demonstrated that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNAi cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi networkderived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity. Thus, we have shown that disruption of LAMP1 expression has a pleiotropic effect on NK cells, causing impairment of trafﬁcking of a critical lytic granule protein, perforin, and slower movement of lytic granules. This results in the inability of NK cells to deliver granzyme B to target cells and inhibition of NK-cell cytotoxicity. Thus, LAMP1 is not only a marker of NK-cell degranulation, but also a crucial component of NK-cell cytotoxicity.

天然杀伤（NK）细胞包括涉及针对微生物病原体和肿瘤的淋巴细胞的子集。由于其细胞毒性能力，NK细胞能够直接消除异常细胞。 NK细胞的裂解颗粒，含有穿孔蛋白和颗粒酶，对于NK细胞细胞毒性是必不可少的，因为它们的释放导致靶细胞细胞凋亡的诱导。裂解颗粒分泌的缺陷与严重的疾病有关，例如淋巴淋巴虫组织细胞增多症和切迪亚克·希加西（Chediak-Higashi），赫尔曼斯基·帕德拉克（Hermansky-Pudlak）或griscelli综合征。关于裂解颗粒释放调节的蛋白质的知识非常有限，并且对NK细胞胞吐作用涉及的颗粒特异性蛋白质机制也很少了解。裂解颗粒具有溶酶体的特征，被称为分泌溶酶体。在溶酶体膜中有许多蛋白质在酸化溶酶体管腔，蛋白质进口，膜融合以及降解产物转运到细胞质中的运输。令人惊讶的是，最丰富的蛋白质，溶酶体相关的膜蛋白（LAMP）1和LAMP2（分别为CD107A和CD107B）的作用是不明确的。在人类中，LAMP2中的突变引起了Danon病，被认为是由异常自噬引起的。 LAMP2缺陷型小鼠还积累了几个组织中的自噬液泡。 LAMP1缺陷型小鼠的表型几乎正常，但LAMP2水平升高，这表明LAMP2可以补偿LAMP1的损失。使用灯耗尽的成纤维细胞的最新研究表明，这两种蛋白质都需要适当的溶酶体传输和有效（自动）吞噬体散糖体融合所必需。 LAMP1也被广泛用作鉴定NK细胞脱粒的标记，因为它在溶酶体与质膜融合后出现在细胞表面上，但是LAMP1在NK细胞生物学中的作用是完全未知的。由于LAMP1是溶酶体膜中最丰富的蛋白质之一，而NK细胞使用其溶酶体诱导靶细胞死亡，因此我们试图确定LAMP1在NK细胞溶解活性中的重要性。因此，该项目的重点是了解溶酶体膜蛋白1，LAMP1在调节人NK细胞细胞毒性中的作用。为了解决这个问题，研究了RNA干扰（RNAI）在人NK细胞系以及原代NK细胞中破坏LAMP1的表达，并研究了LAMP1沉默对裂解颗粒组成，运动，胞吐作用和NK细胞毒性势的影响。我们已经证明，随着Lamp1 RNAi细胞未能向靶细胞递送颗粒酶B，LAMP1沉默会导致NK细胞细胞毒性的抑制。 LAMP1表达的降低会影响裂解颗粒的运动，并导致颗粒中的透明蛋白水平降低，但不会降低颗粒酶B。在LAMP1 RNAi细胞中，在反式高尔基网络衍生囊泡中，更多的细胞蛋白保留在溶酶体室之外。 LAMP1结合伴侣，衔接蛋白1（AP-1）排序复合物的表达的破坏还会导致穿孔囊泡中的穿孔蛋白保留并抑制细胞毒性，这表明AP-1排序复合物与LAMP1在运输囊泡表面上的相互作用对于对裂解液体的运输囊泡表面上的相互作用很重要。我们得出的结论是，在LAMP1缺陷细胞的裂解颗粒中，穿孔蛋白水平降低，结合裂解颗粒的运动性扰动，导致无法将凋亡促进性颗粒的颗粒剂B传递给靶细胞，并抑制NK细胞毒性细胞。因此，我们已经表明，LAMP1表达的破坏对NK细胞具有多效效应，从而导致临界裂解颗粒蛋白，培根和裂解颗粒运动较慢的传统损害。这导致NK细胞无法将颗粒酶B输送到靶细胞并抑制NK细胞细胞毒性。因此，LAMP1不仅是NK细胞脱粒的标记，而且是NK细胞细胞毒性的关键成分。