Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation
| Autor: | Shinichi Hatta, Mayu Otani, Jun Imai, Takahiro Sakai |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Antigen Presentation
General Immunology and Microbiology biology General Chemical Engineering General Neuroscience T cell Endocytic cycle Antigen presentation Histocompatibility Antigens Class I Immunology Cross-presentation Dendritic Cells Endoplasmic Reticulum-Associated Degradation Major histocompatibility complex General Biochemistry Genetics and Molecular Biology Cell biology medicine.anatomical_structure Cross-Priming Antigen medicine biology.protein Humans Antigens Cellular compartment CD8 |
| Zdroj: | Journal of visualized experiments : JoVE. (126) |
| ISSN: | 1940-087X |
| Popis: | Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of naïve CD8(+) T cells and memory CD8(+) T cells for infectious and tumor immunity but also in the inactivation of self-acting naïve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP. |
| Databáze: | OpenAIRE |
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