Dataset Information

Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation.

ABSTRACT: Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE's immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-?, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.

SUBMITTER: Zhang W

PROVIDER: S-EPMC6298919 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

Dataset Information

Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets