Antigen processing is an integral part of cell-mediated immune surveillance and responses that involve MHC- restriction and T cell recognition. Processed antigenic peptides, when presented on cell surface by MHC molecules to T cells, serve as identity tags to be monitored by our immune system. Thus antigen processing and presentation influence the response of an individual to infectious organisms and has been implicated in the susceptibility to diseases and to the development of autoimmunity. One effective way the immune system eliminates virus-infected or malignant cells is to mount a cytotoxic reaction that result in lysis of target cells. This antiviral mechanism generally involves MHC class I molecules to bind peptides processed from viral proteins synthesized within infected cells, and present those foreign antigenic peptides to cytotoxic T lymphocytes (CTLs), which can eliminate virus-infected self-cells and thus eliminate potential sources of new viral production. Therefore, understanding the mechanisms of antigen processing is critical to fight against various pathogens. For an epitope to be recognized by T cells, foreign antigens need to be cut into short peptides with proper sizes in order to form physical complexes with MHC molecules. The precursors of class I antigenic peptides are generated mainly by proteasomes in the cytosol, and are then transported into the lumen of the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Recently, an aminopeptidase inside the ER, named ERAP1, has been identified to be one missing link that trims peptide precursors and generates the final N-termini of class I-restricted epitopes. Our recent success in expressing and purifying active ERAP1, and obtaining crystals of its C-terminal domain (C-dom) in the preliminary crystallization screens, have brought about the exciting possibility of providing high-resolution structural information for this critical enzyme. In this Small Research Project (R03) application, we propose to purify crystallographic quality and quantity of ERAP1 C-dom and complexes for biochemical characterization and high-resolution crystal structure determination.
Crystallographic quality and quantity of ERAP1 C-dom, N-dom, and full-length protein obtained in this proposal will enable biochemical and structural studies of ERAP1 to enhance our understanding of the mechanisms of antigen processing. This information is necessary for the development of protective strategies for biodefense and is thus critical to public health.
| Sui, Lufei; Gandhi, Amit; Guo, Hwai-Chen (2016) Crystal structure of a polypeptide's C-terminus in complex with the regulatory domain of ER aminopeptidase 1. Mol Immunol 80:41-49 |
| Gandhi, Amit; Lakshminarasimhan, Damodharan; Sun, Yixin et al. (2011) Structural insights into the molecular ruler mechanism of the endoplasmic reticulum aminopeptidase ERAP1. Sci Rep 1:186 |
